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| United States Patent Application |
20180346926
|
| Kind Code
|
A1
|
|
Larrinua; Ignacio Mario
; et al.
|
December 6, 2018
|
SYNTHETIC GENES
Abstract
The invention provides synthetic nucleic acid sequences encoding proteins
of interest that are particularly adapted to express well in plants. The
claimed synthetic sequences utilize plant-optimized codons roughly in the
same frequency at which they are utilized, on average, in genes naturally
occurring in the plant species. The invention further includes synthetic
DNA sequence for herbicide tolerance, water and/or heat stress tolerance,
healthy oil modifications and for transformation marker genes and
selectable marker genes are used. DNA construct and transgenic plants
containing the synthetic sequences are taught as are methods and
compositions for using the plants in agriculture.
| Inventors: |
Larrinua; Ignacio Mario; (Indianapolis, IN)
; Merlo; Donald J.; (Carmel, IN)
; Reddy; Avutu S.; (Carmel, IN)
; ThirumalaiswamySekhar; Arvind Kumar; (Zionsville, IN)
; Woosley; Aaron T.; (Fishers, IN)
|
| Applicant: | | Name | City | State | Country | Type |
DOW AGROSCIENCES LLC |
Indianapolis |
IN |
US | | |
|---|
| Family ID:
|
47007465
|
| Appl. No.:
|
16/049169
|
| Filed:
|
July 30, 2018 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 15194925 | Jun 28, 2016 | |
| | 16049169 | | |
| | 13447836 | Apr 16, 2012 | 9427003 |
| | 15194925 | | |
| | 61475921 | Apr 15, 2011 | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
C12Y 113/00 20130101; C07K 14/325 20130101; C12N 9/0083 20130101; C07K 14/415 20130101; C12N 15/8286 20130101; A23L 7/198 20160801; A23D 9/00 20130101; C12N 9/0069 20130101; Y02A 40/146 20180101; C11B 1/00 20130101; A23L 19/10 20160801; C12N 9/0065 20130101; C07K 14/38 20130101; A23L 11/05 20160801; A23L 7/10 20160801; C12Y 111/01015 20130101; C07K 14/195 20130101; C07K 14/37 20130101; A23L 11/00 20160801; C07K 14/43595 20130101 |
| International Class: |
C12N 15/82 20060101 C12N015/82; A23D 9/00 20060101 A23D009/00; C07K 14/415 20060101 C07K014/415; C12N 9/02 20060101 C12N009/02; C07K 14/195 20060101 C07K014/195; C07K 14/37 20060101 C07K014/37; C07K 14/38 20060101 C07K014/38; A23L 19/10 20060101 A23L019/10; A23L 11/00 20060101 A23L011/00; A23L 7/10 20060101 A23L007/10; C12N 9/08 20060101 C12N009/08; C11B 1/00 20060101 C11B001/00; C07K 14/435 20060101 C07K014/435; C07K 14/325 20060101 C07K014/325 |
Claims
1-6. (canceled)
7. A synthetic DNA sequence for expressing a protein of interest in
soybean cells which comprises: a) a codon-optimized DNA sequence encoding
the protein of interest, b) at least one polyadenylation signal sequence
chosen from the group consisting of Class I and Class III wherein; Class
I is chosen from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA,
AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA,
AATTAA, AATACA, and CATAAA; and Class III is chosen from the group
consisting of ATTTTT, TATTTT, TTATTT, TTTATT, TTTTTT, TTTTAT, AATTTT,
TTTTTA, ATATAT, TAATTT, TTAATT, AAATTT, AAATAA, ATATTT, TTTGTT TTGTTT,
ATTATT, ATTTTA, TTTAAT, and TTTTAA, and wherein said codon-optimized DNA
sequence contains at least one polyadenylation signal sequence from Class
III and wherein said synthetic DNA sequence contains fewer Class III
polyadenylation signal sequences than the protein's native DNA sequence
and contains the same number of Class I polyadenylation signal sequences
compared to said native DNA sequence.
8. The synthetic DNA sequence of claim 7 that is substantially devoid of
class III polyadenylation signal sequences.
9. The synthetic DNA sequence of claim 7 that is devoid of class III
polyadenylation signal sequences.
10. The synthetic DNA sequence of claim 7 wherein said synthetic DNA
sequence encodes a native protein selected from the group consisting of
insecticidal proteins, herbicide tolerance proteins, stress
tolerance-related proteins, and oil profile modification proteins.
11. The synthetic DNA sequence of claim 10 wherein said synthetic DNA
sequence encodes an insecticidal protein.
12. The synthetic DNA sequence of claim 10 wherein said synthetic DNA
sequence encodes aryloxyalkanoate dioxygenase 1 protein.
13-14. (canceled)
15. A DNA construct for expression of a protein of interest comprising a
5' non-translated sequence, a coding sequence for a protein of interest,
and a 3' non-translated region, wherein said 5' nontranslated sequence
contains a promoter functional in a plant cell, said coding sequence is
the synthetic DNA coding sequence of claim 7, and wherein said 3'
nontranslated sequence comprises transcription termination sequence and a
polyadenylation signal.
16. (canceled)
17. A transgenic plant containing the synthetic DNA sequence of claim 7.
18. (canceled)
19. A method of controlling pests in grain or seed which comprises
obtaining said grain or seed from plants containing the synthetic DNA of
claim 11.
20-22. (canceled)
23. A composition derived from the transgenic plant of claim 17 wherein
said composition is a commodity product selected from the group
consisting of meal, flour, protein concentrate, or oil.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to provisional application Ser.
No. 61/475,921 filed Apr. 15, 2011, the disclosure of which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] To achieve desired expression levels of heterologous proteins in
transgenic plants it has been found beneficial to alter the native,
sometimes referred to as wild-type or original, DNA coding sequence in
various ways, e.g. so that codon usage more closely matches the codon
usage of the host plant species, and/or so the G+C content of the coding
sequence more closely matches the G+C level typically found in coding
sequences of the host plant species, and/or so that certain sequences
that destabilize mRNA are removed. Expression in plants of Bacillus
thuringiensis (B.t.) crystal protein insect toxins, for example, has been
improved using one or more of these approaches. See, for example, U.S.
Pat. No. 5,380,301, U.S. Pat. No. 5,625,136, U.S. Pat. No. 6,218,188,
U.S. Pat. No. 6,340,593, U.S. Pat. No. 6,673,990, U.S. Pat. No.
7,741,118. Codon degeneracy allows one to make synthetic DNA sequences
that encode a protein of interest using codons that differ from those
used in the original DNA coding sequence.
[0003] In regard to removing sequences that may destabilize mRNA, U.S.
Pat. No. 7,741,118 discloses a list of 16 polyadenylation signal
sequences (column 15, Table II) and calls for reducing the number of such
sequences in synthetic coding sequences that are intended for expression
in plants. The polyadenylation signal sequences listed in U.S. Pat. No.
7,741,118, Table II are listed below in Table 1:
TABLE-US-00001
TABLE 1
Polyadenylation signal sequences listed in
U.S. Pat. No. 7741118, Table II.
1 AATAAA 6 ATACTA 11 ATACAT 16 CATAAA
2 AATAAT 7 ATAAAA 12 AAAATA
3 AACCAA 8 ATGAAA 13 ATTAAA
4 ATATAA 9 AAGCAT 14 AATTAA
5 AATCAA 10 ATTAAT 15 AATACA
[0004] U.S. Pat. No. 7,741,118 also calls for preferably removing the
sequence ATTTA (known as the Shaw-Kamen sequence), because it has been
identified as potentially destabilizing mRNA.
[0005] Contrary to the teaching of U.S. Pat. No. 7,741,118, we have found
that reduction in the number of the polyadenylation signal sequences
identified in Table 1 above is neither necessary nor sufficient to enable
enhanced expression of synthetic genes in plants.
SUMMARY OF THE INVENTION
[0006] Table 2 below identifies 20 potential polyadenylation signal
sequences that occur frequently in maize genes.
TABLE-US-00002
TABLE 2
Potential polyadenylation signal sequences
.English Pound.ound''m maize genes
1 ATATAT 6 TATTTT 11 TAATAA 16 TATTAT
2 TTGTTT 7 TTTTTT 12 ATTTAT 17 TGTTTG
3 TTTTGT 8 ATTTTT 13 TATATT 18 TTATAT
4 TGTTTT 9 TTATTT 14 TTTTAT 19 TGTAAT
5 TATATA 10 TTTATT 15 ATATTT 20 AAATAA
[0007] Table 3 below identifies 20 potential polyadenylation signal
sequences that occur frequently in soybean genes.
TABLE-US-00003
TABLE 3
Potentinl polyadenylation signal sequence
.English Pound.ound''m soybean genes.
1 ATTTTT 6 TTTTAT 11 AAATTT 16 ATATAT
2 TATTTT 7 AATTTT 12 AAATAA 17 ATTATT
3 TTATTT 8 TTTTTA 13 ATATTT 18 ATTTTA
4 TTTATT 9 TAATTT 14 TTTGTT 19 TTTAAT
5 TTTTTT 10 TTAATT 15 TTGTTT 20 TTTTAA
[0008] The present invention provides a synthetic DNA sequence for
expressing a protein of interest in maize cells which comprises: [0009]
a) a codon-optimized DNA sequence encoding the protein of interest,
[0010] b) at least one polyadenylation signal sequence chosen from the
group consisting of Class I and Class II wherein: [0011] Class I is
chosen from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA,
AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA,
AATTAA, AATACA, and CATAAA; and [0012] Class II is chosen from the group
consisting of ATATAT, TTGTTT, TTTTGT, TGTTTT, TATATA, TATTTT, TTTTTT,
ATTTTT, TTATTT, TTTATT, TAATAA, ATTTAT, TATATT, TTTTAT, ATATTT, TATTAT,
TGTTTG, TTATAT, TGTAAT, and AAATAA; and
[0013] wherein said codon-optimized DNA sequence contains-at least one
polyadenylation signal sequence from Class II and wherein said synthetic
DNA sequence contains fewer Class II polyadenylation signal sequences
than the protein's native DNA sequence and contains the same number of
Class I polyadenylation signal sequences compared to the native DNA
sequence.
[0014] The present invention also provides a synthetic DNA sequence for
expressing a protein of interest in soybean cells which comprises:
[0015] a) a codon-optimized DNA sequence encoding the protein of
interest, [0016] b) at least one polyadenylation signal sequence chosen
from the group consisting of Class I and Class III wherein: [0017] Class
I is chosen from the group consisting of AATAAA, AATAAT, AACCAA, ATATAA,
AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT, AAAATA, ATTAAA,
AATTAA, AATACA, and CATAAA; and [0018] Class III is chosen from the group
consisting of ATTTTT, TATTTT, TTATTT, TTTATT, TTTTTT, TTTTAT, AATTTT,
TTTTTA, ATATAT, TAATTT, TTAATT, AAATTT, AAATAA, ATATTT, TTTGTT TTGTTT,
ATTATT, ATTTTA, TTTAAT, and TTTTAA, and
[0019] wherein said codon-optimized DNA sequence contains at least one
polyadenylation signal sequence from Class III and wherein said synthetic
DNA sequence contains fewer Class III polyadenylation signal sequences
than the protein's native DNA sequence and contains the same number of
Class I polyadenylation signal sequences compared to the native DNA
sequence.
[0020] The invention also provides a method of making a synthetic DNA
sequence that encodes a protein of interest which comprises (a) starting
with an amino acid sequence of a protein of interest derived from
naturally occurring polypeptide(s) encoded by native sequence(s) that
comprise at least one polyadenylation signal sequence listed in Table 2,
and (b) making a synthetic DNA sequence that encodes said amino acid
sequence and contains fewer polyadenylation signal sequences listed in
Table 2 compared to the corresponding coding sequence of the native
sequence(s) and contains the same number of polyadenylation signal
sequences listed in Table 1.
[0021] In another embodiment the invention provides a method of making a
synthetic DNA sequence that encodes a protein of interest which comprises
(a) starting with an amino acid sequence of a protein of interest derived
from naturally occurring polypeptide(s) encoded by native sequence(s)
that comprise at least one polyadenylation signal sequence-listed in
Table 3, and (b) making a synthetic DNA sequence that encodes said amino
acid sequence and contains fewer polyadenylation signal sequences listed
in Table 3 compared to the corresponding coding sequence of the native
sequence(s) and contains the same number of polyadenylation signal
sequences listed in Table 1.
[0022] In some embodiments the synthetic DNA sequences provided by the
invention are devoid of the polyadenylation signal sequences listed in
Table 2 and/or Table 3, or the number of polyadenylation signal sequences
identified in Table 2 and/or Table 3 is reduced as much as possible
consistent with maintaining the same number of polyadenylation signal
sequences identified in Table 1 and maintaining the Table 1 sequences in
their original positions in the sequence.
[0023] In some embodiments the synthetic DNA sequences provided by the
invention encode an insecticidal protein, optionally derived from
Bacillus thuringiensis, as well as DNA sequences useful for herbicide
tolerance, water and/or heat stress tolerance, healthy oil modifications
and for transformation marker genes and selectable marker genes.
[0024] The synthetic DNA sequences of the invention may be used in a DNA
construct for expression of a protein of interest, where the construct
comprises a 5' non-translated sequence, a synthetic DNA sequence of the
invention, and a 3' non-translated region, and said 5' non-translated
sequence contains a promoter that functions in plants, and said 3'
non-translated sequence comprises a transcription termination and
polyadenylation signal.
[0025] The invention also provides a transgenic plant containing the
synthetic DNA sequences of the invention.
[0026] Also provided is a method of controlling pests in a plant which
comprises expressing a synthetic DNA sequence of the invention in the
plant where the synthetic DNA sequence encodes an insect toxin, for
example a Bacillus thuringiensis Cry protein.
[0027] Also provided is a method for herbicide tolerance in a plant which
comprises expressing a synthetic DNA sequence of the invention in the
plant where the synthetic DNA sequence encodes a known herbicide
tolerance enzyme, for example the ryloxylkanoate gioxygenase (AAD1) see
WO/2005/107437, or phosphinothricin acetytransferase, or
5-enolpyruvylshikimate-3-phosphate synthase enzymes.
[0028] Also provided is a method for modifying oil profiles in a plant
which comprises expressing one or more synthetic DNA sequences of the
invention in the plant where the synthetic DNA sequence encodes one or
more known enzymes for modifying oil profiles in plants, for example
fatty acid desaturase.
[0029] Also provided is a method for stress tolerance in a plant which
comprises expressing a synthetic DNA sequence of the invention in the
plant where the synthetic DNA sequence encodes known stress tolerance
genes for water and/or heat stress, for example the stress associated
protein (SAP1); US Patent Publication No: 2010/0275327, and 1-Cys
peroxiredoxin (Perl) proteins (Mowla et al., 2002, Planta 215:716-726).
[0030] Also provided is a method adding reporter genes in a plant which
comprises expressing a synthetic DNA sequence of the invention in the
plant where the synthetic DNA sequence encodes a known transformation
marker protein functional in plants, for example green fluorescence
protein (GFP) or beta glucuronidase enzyme.
[0031] Also provided is a method of controlling pests in grain or seed
which comprises obtaining said grain or seed from plants containing a
synthetic gene of the invention that expresses an insect toxin, and a
method of controlling pests in meal or flour which comprises obtaining
said meal or flour from grain containing a synthetic gene of the
invention that expresses an insect toxin.
[0032] Also provided is a composition derived from transgenic plants
containing synthetic DNA of the invention wherein said composition is a
commodity product selected from the group consisting of meal, flour,
protein concentrate, or oil.
[0033] In some cases the number of polyadenylation signals listed in Table
1 can be maintained in synthetic DNA sequences of the invention by
deleting occurrences of AATAAA and substituting another polyadenylation
signal sequence listed in Table 1. This is exemplified in Example 1, SEQ
ID NO:5.
DESCRIPTION OF THE SEQUENCES
[0034] SEQ ID NO:1 is the native DNA sequence encoding Bacillus
thuringiensis Cry1Fa core toxin.
[0035] SEQ ID NO:2 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0036] SEQ ID NO:3 is a synthetic DNA sequence encoding Bacillus
thuringiensis Cry1Fa core toxin using codons optimized for maize and
Table 1 sequences are maintained.
[0037] SEQ ID NO:4 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0038] SEQ ID NO:5 is a synthetic DNA sequence in accordance with the
invention encoding-Bacillus thuringiensis Cry1Fa core toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0039] SEQ ID NO 6 is Bacillus thuringiensis Cry1Fa core toxin sequence.
[0040] SEQ ID NO:7 is the native DNA sequence encoding Bacillus
thuringiensis Cry34Ab1 toxin.
[0041] SEQ ID NO:8 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0042] SEQ ID NO:9 is a synthetic DNA sequence encoding Bacillus
thuringiensis Cry34Ab1 toxin using codons optimized for maize and Table 1
sequences are maintained.
[0043] SEQ ID NO: 10 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0044] SEQ ID NO: 11 is a synthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry34Ab1 toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0045] SEQ ID NO: 12 is Bacillus thuringiensis Cry34Ab1 toxin sequence.
[0046] SEQ ID NO: 13 is the native DNA sequence encoding Bacillus
thuringiensis Cry35Ab1 toxin.
[0047] SEQ ID NO:14 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0048] SEQ ID NO: 15 is a synthetic DNA sequence encoding Bacillus
thuringiensis Cry35Ab1 toxin using codons optimized for maize and Table 1
sequences are maintained
[0049] SEQ ID NO:16 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0050] SEQ ID NO: 17 is a synthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry35Ab1 toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0051] SEQ ID NO:18 is Bacillus thuringiensis Cry35Ab1 toxin sequence.
[0052] SEQ ID NO:19 is the native DNA sequence encoding Bacillus
thuringiensis Cry1Ab1 core toxin.
[0053] SEQ ID NO:20 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0054] SEQ ID NO:21 is a synthetic DNA sequence encoding Bacillus
thuringiensis Cry1Ab1 core toxin using codons optimized for maize and
Table 1 sequences are maintained.
[0055] SEQ ID NO:22 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0056] SEQ ID NO:23 is a synthetic DNA sequence in accordance with the
invention encoding-Bacillus thuringiensis Cry1Ab1 core toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0057] SEQ ID NO:24 is Bacillus thuringiensis Cry1Ab1 core toxin sequence.
[0058] SEQ ID NO:25 is the native DNA sequence encoding Bacillus
thuringiensis CryICa core toxin.
[0059] SEQ ID NO:26 is encoding Bacillus thuringiensis CryICa core toxin
sequence.
[0060] SEQ ID NO:27 is a synthetic DNA sequence encoding Bacillus
thuringiensis CryICa core toxin using codons optimized for maize and
Table 1 sequences are maintained.
[0061] SEQ ID NO:28 is encoding Bacillus thuringiensis CryICa core toxin
sequence.
[0062] SEQ ID NO:29 is a synthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis CryICa core toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0063] SEQ ID NO:30 is encoding Bacillus thuringiensis CryICa core toxin
sequence.
[0064] SEQ ID NO:31 is the native DNA sequence encoding Bacillus
thuringiensis Cry6Aa toxin.
[0065] SEQ ID NO:32 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0066] SEQ ID NO:33 is a synthetic DNA sequence encoding Bacillus
thuringiensis Cry6Aa toxin using codons optimized for maize and Table 1
sequences are maintained.
[0067] SEQ ID NO:34 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0068] SEQ ID NO:35 is a synthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry6Aa toxin using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0069] SEQ ID NO:36 is Bacillus thuringiensis Cry6Aa toxin sequence.
[0070] SEQ ID NO:37 is the native DNA sequence encoding Sphingobiurn
herbicidovorans AAD1 protein.
[0071] SEQ ID NO:38 is Sphingobiurn herbicidovorans AADI protein sequence.
[0072] SEQ ID NO:39 is a synthetic DNA sequence encoding Sphingobiurn
herbicidovorans AAD1 protein using codons optimized for maize and Table 1
& Table 2 sequences are maintained.
[0073] SEQ ID NO:40 is Sphingobiurn herbicidovorans AADI protein sequence.
[0074] SEQ ID NO:41 is a synthetic DNA sequence in accordance with the
invention encoding Sphingobiurn herbicidovorans AAD1 protein using codons
optimized for maize and with sequences identified in Table 2 removed and
Table 1 sequences are maintained.
[0075] SEQ ID NO:42 is Sphingobiurn herbicidovorans AADI protein sequence.
[0076] SEQ ID NO:43 is the native DNA sequence encoding Aspergillus
nidulans delta-9 fatty acid desaturase protein.
[0077] SEQ ID NO:44 is Aspergillus nidulans delta-9 fatty acid desaturase
protein sequence.
[0078] SEQ ID NO:45 is a synthetic DNA sequence encoding Aspergillus
nidulans delta-9 fatty acid desaturase protein using codons optimized for
maize and Table 1 & Table 2 sequences are maintained.
[0079] SEQ ID NO:46 is Aspergillus nidulans delta-9 fatty acid desaturase
protein sequence.
[0080] SEQ ID NO:47 is a synthetic DNA sequence in accordance with the
invention encoding Aspergillus nidulans delta-9 fatty acid desaturase
protein using codons optimized for maize and with sequences identified in
Table 2 removed and Table 1 sequences are maintained.
[0081] SEQ ID NO:48 is Aspergillus nidulans delta-9 fatty acid desaturase
protein.
[0082] SEQ ID NO:49 is the native DNA sequence encoding Xerophyta viscosa
SAPI protein.
[0083] SEQ ID NO:50 is Xerophyta viscosa SAPI protein sequence.
[0084] SEQ ID NO:51 is a synthetic DNA sequence encoding Xerophyta viscosa
SAPI protein using codons optimized for maize and Table 1 & Table 2
sequences are maintained
[0085] SEQ ID NO:52 is Xerophyta viscosa SAPI protein sequence.
[0086] SEQ ID NO:53 is a synthetic DNA sequence in accordance with the
invention encoding Xerophyta viscosa SAP1 protein using codons optimized
for maize and with sequences identified in Table 2 removed and Table I
sequences are maintained
[0087] SEQ ID NO:54 is Xerophyta viscosa SAPI protein sequence.
[0088] SEQ ID NO:55 is the native DNA sequence encoding Aequorea victoria
GFPI protein.
[0089] SEQ ID NO:56 is Aequorea victoria GFPI protein sequence.
[0090] SEQ ID NO:57 is a synthetic DNA sequence encoding Aequorea victoria
GFPI protein using codons optimized for maize and Table I & Table 2
sequences are maintained.
[0091] SEQ ID NO:58 is Aequorea victoria GFPI protein sequence.
[0092] SEQ ID NO:59 is a synthetic DNA sequence in accordance with the
invention encoding Aequorea victoria GFPI protein using codons optimized
for maize and with sequences identified in Table 2 removed and Table I
sequences are maintained.
[0093] SEQ ID NO:60 is Aequorea victoria GFPI protein sequence.
[0094] SEQ ID NO:6I is the native DNA sequence encoding Leptosphaeria
nodorum delta-9 fatty acid desaturase protein.
[0095] SEQ ID NO:62 is Leptosphaeria nodorum delta-9 fatty acid desaturase
protein sequence.
[0096] SEQ ID NO:63 is a synthetic DNA sequence encoding Leptosphaeria
nodorum delta-9 fatty acid desaturase protein using codons optimized for
maize and Table I & Table 2 sequences are maintained
[0097] SEQ ID NO:64 is Leptosphaeria nodorum delta-9 fatty acid desaturase
protein sequence.
[0098] SEQ ID NO:65 is a synthetic DNA sequence in accordance with the
invention encoding Leptosphaeria nodorum delta-9 fatty acid desaturase
protein using codons optimized for maize and with sequences identified in
Table 2 removed and Table I sequences are maintained
[0099] SEQ ID NO:66 is Leptosphaeria nodorum delta-9 fatty acid desaturase
protein sequence.
[0100] SEQ ID NO:67 is the native DNA sequence encoding Xerophyta viscosa
PERI protein.
[0101] SEQ ID NO:68 is Xerophyta viscosa PERI protein sequence.
[0102] SEQ ID NO:69 is a synthetic DNA sequence encoding Xerophyta viscosa
PERI protein using codons optimized for maize and Table I & Table 2
sequences are maintained.
[0103] SEQ ID NO:70 is Xerophyta viscosa PERI protein sequence.
[0104] SEQ ID NO:7I is a synthetic DNA sequence in accordance with the
invention encoding-Xerophyta viscosa PERI protein using codons optimized
for maize and with sequences identified in Table 2 removed and Table I
sequences are maintained.
[0105] SEQ ID NO:72 is Xerophyta viscosa PERI protein sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0106] The invention provides synthetic nucleic acid sequences encoding
proteins of interest. The synthetic coding sequences are particularly
adapted for use in expressing the proteins of interest in transgenic
plants.
[0107] A protein of interest is any protein or polypeptide that occurs in
nature, or any naturally occurring variant including but not limited to
processed forms of such proteins. The protein of interest also may be a
protein formed by combining portions or fragments of more than one
naturally occurring protein such as by mixing and matching functional
protein domains.
[0108] A preferred group of proteins of interest is one in which the
resulting phenotype is an agronomic trait or reporter protein useful for
creating agronomic traits. These include but are not limited to
resistance to insects, tolerance to herbicides, tolerance to water and/or
heat stress, and oil profile modification.
[0109] A more preferred group of proteins of interest is one in which the
resulting phenotype is an agronomic trait. Another preferred group is one
in which the resulting phenotype provides herbicide tolerance. Another
preferred group is one in which the resulting phenotype provides stress
tolerance. Another preferred group is one in which the resulting
phenotype provides a modified oil profile for healthier food. A more
highly preferred group is one in which the protein of interest is a Cry
protein that provides insect resistance.
[0110] The native/wild-type DNA sequences encoding the protein of interest
must be identified and analyzed to determine whether polyadenylation
signal sequences listed in Tables I and 2 and/or 3 are present. In
accordance with the invention, for coding sequences intended for use in
maize, the number of polyadenylation signal sequences listed in Table 2
is reduced compared to the number present in the native sequence. For
coding sequences intended for use in soybean, the number of
polyadenylation signal sequences listed in Table 3 is reduced. It is very
important to remove the polyadenylation signal sequences listed in Tables
2 and 3, particularly where they occur in nested multimeric form.
[0111] In addition to removing polyadenylation signal sequences listed in
Tables 2 and 3, it may be desirable to remove occurrences of the
Shaw-Kamen sequence, ATTTA.
[0112] In addition to removing polyadenylation signal sequences and
Shaw-Kamen sequences, we prefer to build synthetic DNA coding sequences
that utilize codons roughly in the same frequency at which they are
utilized, on average, in genes naturally occurring in the plant species
in which the synthetic DNA sequence will be used. Table 4 gives suitable
target percentages for codon usage in synthetic genes intended for use in
various specific crops as well as for use in dicots generally or plants
generally.
TABLE-US-00004
TABLE 4
Target rescaled codon compositions of synthetic plant genes.
Maize Soybean Maize Soybean
Amino Acid Codon % % Amino Acid Codon % %
ALA (A) GCA 18.0 33.1 LEU (L) CTA 0 0
GCC 34.0 24.5 CTC 29.9 22.4
GCG 24.0 0 CTG 33.3 16.3
GCT 24.0 42.3 CTT 19.5 31.5
ARG (R) AGA 18.8 36.0 TTA 0 0
AGG 32.5 32.2 TTG 17.2 29.9
CGA 0 0 LYS (K) AAA 22.0 42.5
CGC 30.0 15 AAG 78.0 57.5
CGTG 18.8 0 MET (M) ATG 100 100
CGT 0 16.9 PHE (F) TTC 71.0 49.2
ASN (N) AAC 68.0 50.0 TTT 29.0 50.8
AAT 32.0 50.0 PRO (P) CCA 26.0 39.8
ASP (D) GAC 63.0 38.1 CCC 24.0 20.9
GAT 37.0 61.9 CCG 28.0 0.0
CYS (C) TGC 68.0 50.0 CCT 22.0 39.3
TGT 32.0 50.0 SER (S) AGC 25.3 16.0
END TAA 0 0 AGT 0.0 18.2
TAG 0 0 TCA 17.6 21.9
TGA 100 100 TCC 25.3 18.0
GLN (Q) CAA 38.0 55.5 TCG 15.4 0
CAG 62.0 44.5 TCT 16.5 25.8
GLU (E) GAA 29.0 50.5 THR (T) ACA 21.0 32.4
GAG 71.0 49.5 ACC 37.0 30.2
GLY (G) GGA 19.0 31.9 ACG 22.0 0.0
GGC 42.0 19.3 ACT 20.0 37.4
GGG 19.0 18.4 TRP (W) TGG 100 100
GGT 20.0 30.4 TYR (Y) TAC 73.3 48.2
HIS (H) CAC 62.0 44.8 TAT 27.0 51.8
CAT 38.0 55.2 VAL (V) GTA 0 11.5
ILE (I) ATA 14.0 23.4 GTC 34.8 17.8
ATC 58.0 29.9 GTG 42.4 32.0
ATT 28.0 46.7 GTT 22.8 38.7
Transgenic Plants
[0113] A preferred embodiment of the subject invention is the
transformation of plants with genes encoding insect toxins. The
transformed plants that express insect toxin genes are resistant to
attack by an insect target pest by virtue of the presence of controlling
amounts of the subject insecticidal protein or its variants in the cells
of the transformed plant. By incorporating genetic material that encodes
the insecticidal properties of the B.t. insecticidal toxins into the
genome of a plant eaten by a particular insect pest, the adult or larvae
die after consuming the food plant. Numerous members of the
monocotyledonous and dicotyledonous classifications have been
transformed. Transgenic agronomic crops as well as fruits and vegetables
are of commercial interest. Such crops include but are not limited to
maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat,
cotton, peanuts, tomatoes, potatoes, and the like. Several techniques
exist for introducing foreign genetic material into plant cells, and for
obtaining plants that stably maintain and express the introduced gene.
Such techniques include acceleration of genetic material coated onto
microparticles directly into cells (U.S. Pat. No. 4,945,050 and U.S. Pat.
No. 5,141,131). Plants may be transformed using Agrobacterium technology,
see U.S. Pat. No. 5,177,010, European Patent No. EP131624B1, European
Patent No. EP159418B1, European Patent No. EP17611281, U.S. Pat. No.
5,149,645, EP120516B1, U.S. Pat. No. 5,464,763, U.S. Pat. No. 4,693,976,
European Patent No. EP116718B1, European Patent No. EP290799B1, European
Patent No. EP320500B1, European Patent No. EP604662B1, U.S. Pat. No.
7,060,876, U.S. Pat. No. 6,037,526, U.S. Pat. No. 6,376,234, European
Patent No. EP292435B1, U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174,
U.S. Pat. No. 4,762,785, U.S. Pat. No. 5,608,142, and U.S. Pat. No.
5,159,135. Other transformation technology includes WHISKERS.TM.
technology, see U.S. Pat. No. 5,302,523 and U.S. Pat. No. 5,464,765.
Electroporation technology has also been used to transform plants, see
WO1987006614, U.S. Pat. No. 5,472,869, U.S. Pat. No. 5,384,253,
WO199209696, U.S. Pat. No. 6,074,877, WO1993021335, and U.S. Pat. No.
5,679,558. In addition to numerous technologies for transforming plants,
the type of tissue which is contacted with the foreign genes may vary as
well. Such tissue would include but would not be limited to embryogenic
tissue, callus tissue type I and type II, hypocotyl, meristem, and the
like. Almost all plant tissues may be transformed during
dedifferentiation using appropriate techniques within the skill of an
artisan.
[0114] Known techniques of inserting DNA into plants include
transformation with T-DNA delivered by Agrobacterium tumefuciens or
Agrobacterium rhizogenes as the transformation agent. The use of
T-DNA-containing vectors for the transformation of plant cells has been
intensively researched and sufficiently described in European Patent No.
EP12051681; Lee and Gelvin (2008) Plant Physiol. 146:325-332; Fraley et
al (1986) Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J.
4:277-284; and is well established in the field. Additionally, fusion of
plant protoplasts with liposomes containing the DNA to be delivered,
direct injection of the DNA, biolistics transformation (microparticle
bombardment), or electroporation, as well as other possible methods, may
be employed.
[0115] Once the inserted DNA has been integrated into the plant genome, it
is relatively stable throughout subsequent generations. The vector used
to transform the plant cell normally contains a selectable marker gene
encoding a protein that confers on the transformed plant cells resistance
to a herbicide or an antibiotic, such as Bialaphos, Kanamycin. G418,
Bleomycin, or Hygromycin, inter alia. The individually employed
selectable marker gene should accordingly permit the selection of
transformed cells while the growth of cells that do not contain the
inserted DNA is suppressed by the selective compound.
[0116] In a preferred embodiment of the subject invention, plants are
transformed with genes wherein the codon usage of the protein coding
region has been optimized for plants. See, for example, U.S. Pat. No.
5,380,831. Also, advantageously, plants encoding a truncated toxin, e.g.
a functional protein domain, maybe used. The truncated toxin typically
encodes about 55% to about 80% of the native full length toxin. Methods
for creating synthetic B.t. genes for use in plants are known in the art
(Stewart 2007, Frontiers in Drug Design and Discovery 1:297-341).
[0117] Regardless of transformation technique, the gene is preferably
incorporated into a gene transfer vector adapted to express the protein
of interest in the plant cell by including in the vector a plant
promoter. In addition to plant promoters, promoters from a variety of
sources can be used efficiently in plant cells to express foreign genes.
For example, promoters of bacterial origin, such as the octopine synthase
promoter, the nopaline synthase promoter, the mannopine synthase
promoter, promoters of viral origin, such as the 35S and 19S promoters of
cauliflower mosaic virus (CaMV), and the like may be used. Plant-derived
promoters include, but are not limited to ribulose-1,6-bisphosphate
(RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter,
phaseolin promoter, ADH (alcohol dehydrogenase) promoter, heat-shock
promoters, ADF (actin depolymerization factor) promoter, and tissue
specific promoters. Promoters may also contain certain enhancer sequence
elements that may improve the transcription efficiency. Typical enhancers
include but are not limited to ADHI-intron 1 and ADHI-intron 6.
Constitutive promoters may be used. Constitutive promoters direct
continuous gene expression in nearly all cells types and at nearly all
times (e.g. actin, ubiquitin, CaMV 35S). Tissue specific promoters are
responsible for gene expression in specific cell or tissue types, such as
the leaves or seeds (e.g. zein, oleosin, napin, ACP (Acyl Carrier
Protein)), and these promoters may also be used. Promoters may also be
used that are active during a certain stage of the plants' development as
well as active in specific plant tissues and organs. Examples of such
promoters include but are not limited to promoters that are root
specific, pollen-specific, embryo specific, corn silk specific, cotton
fiber specific, seed endosperm specific, phloem specific, and the like.
[0118] Under certain circumstances it may be desirable to use an inducible
promoter. An inducible promoter is responsible for expression of genes in
response to a specific signal, such as: physical stimulus (e.g. heat
shock genes); light (e.g. RUBP carboxylase); hormone (e.g.
glucocorticoid); antibiotic (e.g. tetracycline), metabolites; and stress
(e.g. drought). Other desirable transcription and translation elements
that function in plants may be used, such as 5' untranslated leader
sequences, RNA transcription termination sequences and poly-adenylate
addition signal sequences. Numerous plant-specific gene transfer vectors
are known to the art.
[0119] Transgenic crops containing insect resistance (IR) traits are
prevalent in corn and cotton plants throughout North America, and usage
of these traits is expanding globally. Commercial transgenic crops
combining IR and herbicide tolerance (HT) traits have been developed by
numerous seed companies. These include combinations of IR traits
conferred by B.t. insecticidal proteins and HT traits such as tolerance
to Acetolactate Synthase (ALS) inhibitors such as sulfonylureas,
imidazolinones, triazolopyrimidine, sulfonanilides, and the like,
Glutamine Synthetase (GS) inhibitors such as Bialaphos, glufosinate, and
the like, 4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such as
mesotrione, isoxaflutole, and the like,
5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such as
glyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase)
inhibitors such as haloxyfop, quizalofop, diclofop, and the like. Other
examples are known in which transgenically provided proteins provide
plant tolerance to herbicide chemical classes such as phenoxy acids
herbicides and pyridyloxyacetates auxin herbicides (see WO2007053482), or
phenoxy acids herbicides and aryloxyphenoxypropionates herbicides (see US
Patent Application No. 20090093366). The ability to control multiple pest
problems through IR traits is a valuable commercial product concept, and
the convenience of this product concept is enhanced if insect control
traits and weed control traits are combined in the same plant. Further,
improved value may be obtained via single plant combinations of IR traits
conferred by a B.t. insecticidal protein such as that of the subject
invention with one or more additional HT traits such as those mentioned
above, plus one or more additional input traits (e.g. other insect
resistance conferred by B.t.-derived or other insecticidal proteins,
insect resistance conferred by mechanisms such as RNAi and the like,
nematode resistance, disease resistance, stress tolerance, improved
nitrogen utilization, and the like), or output traits (e.g high oils
content, healthy oil composition, nutritional improvement, and the like).
Such combinations may be obtained either through conventional breeding
(breeding stack) or jointly as a novel transformation event involving the
simultaneous introduction of multiple genes (molecular stack or
co-transformation). Benefits include the ability to manage insect pests
and improved weed control in a crop plant that provides secondary
benefits to the producer and/or the consumer. Thus, the subject invention
can be used in connection with a variety of traits to provide a complete
agronomic package of improved crop quality with the ability to flexibly
and cost effectively control any number of agronomic issues.
[0120] All patents, patent applications, provisional applications, and
publications referred to or cited herein are incorporated by reference in
their entirety to the extent they are not inconsistent with the explicit
teachings of this specification. Unless specifically indicated or
implied, the terms "a", "an", and "the" signify "at least one" as used
herein. By "isolated" applicants mean that the nucleotide or polypeptide
molecules have been removed from their native environment and have been
placed in a different environment by the hand of man.
[0121] Embodiments of the present invention are further defined in the
following Examples. It should be understood that these Examples are given
by way of illustration only. From the above discussion and these
Examples, one skilled in the art can ascertain the essential
characteristics of this invention, and without departing from the spirit
and scope thereof can make various changes and modifications of the
embodiments of the invention to adapt it to various usages and
conditions. Thus, various modifications of the embodiments of the
invention, in addition to those shown and described herein, will be
apparent to those skilled in the art from the foregoing description. Such
modifications are also intended to fall within the scope of the appended
claims.
[0122] All percentages are by weight and all solvent mixture proportions
are by volume unless otherwise noted. All temperatures are in degrees
Celsius.
Example 1
Synthetic Coding Region Encoding Bacillus thuringiensis Cry1Fa Core Toxin
[0123] Comparative Sequence. The native DNA sequence encoding the Cry1Fa
core toxin is given in SEQ ID NO: 1. This sequence was analyzed to
determine which sequences identified in Table 1 are present in SEQ ID NO:
1 and their locations. The amino acid sequence encoded by SEQ ID NO: 1
was then reverse translated using the target codon frequencies given in
the column of Table 4 for synthetic genes to be used in maize. The
resulting DNA sequence was analyzed and codons were changed where
necessary to remove unwanted open reading frames and remove unwanted
restriction sites, and restore sequences identified in Table 1. The amino
acid sequence encoded by SEQ ID NO: 1 was preserved. The resulting DNA
sequence is given in SEQ ID NO:3.
[0124] SEQ ID NO:3 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the same number of sequences identified in Table 1. The
resulting sequence, which embodies the present invention, is given in SEQ
ID NO:5. Table 5 shows that the number and locations of polyadenylation
signal sequences identified in Table 1 are maintained in SEQ ID NO:5,
with the exception that the two occurrences of AATAAA, one at nt 426 and
one at nt 582, in SEQ ID NO:1 were replaced with AATCAA, which maintains
the number and location of polyadenylation signal sequences identified in
Table 1, but substitutes a less problematic sequence for each of the two
AATAAA sequences. Table 6 shows that the number of polyadenylation signal
sequences identified in Table 2 are reduced in SEQ ID NO:5. Because there
is overlap in the sequences identified in Tables 2 and 3 (sequences 1, 2,
6, 7, 8, 9, 10, 14, 13, and 20 in Table 2 correspond to sequences 16, 15,
2, 5, 1, 3, 4, 6, 13, and 12, respectively, in Table 3) it is also true
that the number of polyadenylation signal sequences identified in Table 3
are reduced in SEQ ID NO:5.
[0125] The synthetic coding region of SEQ ID NO:5 was optimized for
expression in maize.
[0126] A construct for use in expressing the synthetic coding region of
SEQ ID NO:5 is made by combining the synthetic coding region of SEQ ID
NO:5 with a 5' non-translated region comprising a promoter that functions
in plant cells and a 3' non-translated region comprising a transcription
termination and polyadenylation sequence.
[0127] In one embodiment of such a construct, production of the primary
mRNA transcript comprising SEQ ID NO:5 was driven by a copy of a maize
ubiquitin1 promoter with its native intron1 (U.S. Pat. No. 5,510,474). A
fragment comprising a 3' untranslated region from a maize peroxidase 5
gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984) was used to terminate
transcription. A binary plant transformation plasmid, pDAB1 11440,
containing the aforementioned gene expression cassette, was constructed
and utilized in the production of transgenic maize plants. Plasmid pDAB1
11440 further comprises a herbicide resistance gene comprising a coding
region for aryloxyalknoate dioxygenase (AAD-1 v3; U.S. Pat. No.
7,838,733(82), and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A.
107:20240-5) under the transcriptional control of a sugarcane bacilliform
badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol.
39:1221-30). A fragment comprising a 3' untranslated region from a maize
lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) was used to terminate
transcription.
TABLE-US-00005
TABLE 5
Table 1 sequences found in the native Cry1Fa core toxin coding region
(SEQ ID NO: 1) and in the redesigned version (SEQ ID NO: 5)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Fa Native Cry1Fa redesigned Cry1Fa redesigned Cry1Fa
Table 1 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 5) (SEQ ID NO: 5)
1 AATAAA 2 426; 582 0 NA*
2 AATAAT 5 7; 46; 358; 430; 562 5 7; 46; 358; 430; 562
3 AACCAA 0 NA 0 NA
4 ATATAA 1 1520 1 1520
5 AATCAA 2 19; 628 4 19; 426; 582; 628
6 ATACTA 1 1508 1 1508
7 ATAAAA 0 NA 0 NA
8 ATGAAA 2 314; 1211 2 314; 1211
9 AAGCAT 0 NA 0 NA
10 ATTAAT 2 579; 1690 2 579; 1690
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 2 66; 1266 2 66; 1266
14 AATTAA 2 368; 779 2 368; 779
15 AATACA 3 400; 1369; 1693 3 400; 1369; 1693
16 CATAAA 0 NA 0 NA
Total 22 22
*NA = Not Applicable
TABLE-US-00006
TABLE 6
Table 2 sequences found in the native Cry1Fa core toxin coding region
(SEQ ID NO: 1) and in the redesigned version (SEQ ID NO: 5)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Fa Native Cry1Fa redesigned Cry1Fa redesigned Cry1Fa
Table 2 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 1) (SEQ ID NO: 1) (SEQ ID NO: 5) (SEQ ID NO: 5)
1 ATATAT 1 104 0 NA*
2 TTGTTT 3 39; 12; 907 0 NA
3 TTTTGT 1 1089 0 NA
4 TGTTTT 2 1086; 1334 0 NA
5 TATATA 1 1771 0 NA
6 TATTTT 0 NA 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 1 1615 0 NA
9 TTATTT 2 172; 217 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 4 357; 416; 561; 581 0 NA
12 ATTTAT 3 319; 497; 793 0 NA
13 TATATT 1 322 0 NA
14 TTTTAT 3 192; 464; 1063 0 NA
15 ATATTT 0 NA 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 2 613; 908 0 NA
18 TTATAT 2 321; 1770 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 2 45; 429 0 NA
Total 28 0 NA
*NA = Not Applicable
Example 2
Synthetic Coding Region Encoding Bacillus thuringiensis Cry34A Toxin
[0128] Comparative Sequences. The native DNA sequence encoding the Cry34A
toxin is given in SEQ ID NO:7. This sequence was analyzed to determine
which sequences identified in Table 1 are present in SEQ ID NO:7 and
their locations. The native DNA sequence was translated into the
corresponding amino acid sequence using the standard genetic code. The
amino acid sequence encoded by SEQ ID NO:7 was then reverse translated
using the target codon frequencies given in the column of Table 7 for
synthetic genes to be used in maize. The resulting DNA sequence was
analyzed and codons were changed where necessary to remove unwanted open
reading frames and remove unwanted restriction sites, and restore all
sequences identified in Table 1. The amino acid sequence encoded by SEQ
ID NO:7 was preserved. The resulting DNA sequence is given in SEQ ID
NO:9. DNA having the sequence of SEQ ID NO:9 is synthesized.
[0129] SEQ ID NO:9 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the same number of sequences identified in Table 1. The
resulting sequence, which embodies the present invention, is given in SEQ
ID NO: 11. Table 7 shows that the number and locations of polyadenylation
signals sequences identified in Table 1 are maintained in SEQ ID NO: 11.
Table 8 shows that the number of polyadenylation signal sequences
identified in Tables 2 and 3 are reduced in SEQ ID NO:5.
[0130] DNA of SEQ ID NO:5 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:1 and SEQ ID NO:3.
[0131] The synthetic coding region of SEQ ID NO:5 was optimized for
expression in maize.
[0132] A construct for use in expressing the synthetic coding region of
SEQ ID NO:5 is made by combining the synthetic coding region of SEQ ID
NO:5 with a 5' non-translated region comprising a promoter that functions
in plant cells and a 3' non-translated region comprising a transcription
termination and polyadenylation sequence.
TABLE-US-00007
TABLE 7
Table 1 sequences found in the native Cry34Ab1 coding region
(SEQ ID NO: 7) and the redesigned version (SEQ ID NO: 11)
No. Sites in nt Location in No. Sites in nt Location in
native Cry34Ab1 native Cry34Ab1 redesigned redesigned
Table 1 sequence sequence Cry34Ab1 sequence Cry34Ab1 sequenc
Sequence (SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 11) (SEQ ID NO: 11)
1 AATAAA 2 247; 268 2 247; 268
2 AATAAT 1 31 1 31
3 AACCAA 0 NA* 0 NA
4 ATATAA 0 NA 0 NA
5 AATCAA 2 146; 310 2 146; 310
6 ATACTA 1 329 1 329
7 ATAAAA 1 65 1 65
8 ATGAAA 1 281 1 281
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 1 47 1 47
12 AAAATA 0 NA 0 NA
13 ATTAAA 1 127 1 127
14 AATTAA 1 126 1 126
15 AATACA 0 NA 0 NA
16 CATAAA 1 361 1 361
Total 12 12
*NA = Not Applicable
TABLE-US-00008
TABLE 8
Table 2 sequences found in the native Cry34Ab1 coding region
(SEQ ID NO: 7) and in the redesigned version (SEQ ID NO: 11)
No. Sites in nt Location in No. Sites in nt Location in
native Cry34Ab1 native Cry34Ab1 redesigned redesigned
Table 2 sequence sequence Cry34Ab1 sequence Cry34Ab1 sequenc
Sequence (SEQ ID NO: 7) (SEQ ID NO: 7) (SEQ ID NO: 11) (SEQ ID NO: 11)
1 ATATAT 1 181 0 NA*
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 1 180 0 NA
6 TATTTT 1 220 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 2 33; 246 2 33; 246
12 ATTTAT 0 NA 0 NA
13 TATATT 2 182; 218 0 NA
14 TTTTAT 1 156 0 NA
15 ATATTT 1 219 0 NA
16 TATTAT 1 184 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 1 217 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 1 30 1 NA
Total 12 3
*NA = Not Applicable
Example 3
Synthetic Coding Region Encoding Bacillus thuringiensis Cry35Ab1 Toxin
[0133] Comparative Sequences. The native DNA sequence encoding the
Cry35Ab1 toxin is given in SEQ ID NO: 13. This sequence was analyzed to
determine which sequences identified in Table 1 are present in SEQ ID NO:
13 and their locations. The amino acid sequence encoded by SEQ ID NO: 13
was then reverse translated using the target codon frequencies given in
the column of Table 4 for synthetic genes to be used in maize. The
resulting DNA sequence was analyzed and codons were changed where
necessary to remove unwanted open reading frames and remove unwanted
restriction enzyme recognition sites, while maintaining all sequences
identified in Table 1. The amino acid sequence encoded by SEQ ID NO: 13
was preserved. The resulting DNA sequence is given in SEQ ID NO: 15. This
sequence will be synthesized and used for comparison with a synthetic
coding region designed in accordance with the invention.
[0134] SEQ ID NO: 15 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the same number of sequences identified in Table 1, except
that two of the occurrences of AATAAA, one at nt 228 and one at nt 276 of
SEQ ID NO:8 were changed to AATCAA. The resulting sequence, which
embodies the present invention, is given in SEQ ID NO: 17. Table 9 shows
that the number and location of polyadenylation signal sequences
identified in Table 1 are maintained in SEQ ID NO: 17. Table 10 shows
that the number of polyadenylation signal sequences identified in Tables
2 and 3 are reduced in SEQ ID NO:17 compared to SEQ ID NO:13.
[0135] DNA of SEQ ID NO:17 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:13 and SEQ ID NO:15.
[0136] The synthetic coding region of SEQ ID NO: 17 was optimized for
expression in maize.
[0137] A construct for use in expressing the synthetic coding region of
SEQ ID NO: 17 is made by combining the synthetic coding region of SEQ ID
NO: 17 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription termination and polyadenylation sequence.
TABLE-US-00009
TABLE 9
Table 1 sequences found in the native Cry35Ab1 coding region
(SEQ ID NO: 13) and in the redesigned version (SEQ ID NO: 17)
No. Sites in nt Location in No. Sites in nt Location
native Cry native Cry35Ab1 redesigned in redesigned
Table 1 35 Ab1 sequence sequence Cry35Ab1 sequence Cry35Ab1 sequence
Sequence (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 17) (SEQ ID NO: 17)
1 AATAAA 5 13; 100; 228; 276; 810 3 13; 100; 810
2 AATAAT 4 193; 217; 385; 864 4 193; 217; 385; 864
3 AACCAA 0 NA* 0 NA
4 ATATAA 1 966 1 966
5 AATCAA 3 394; 750; 914 5 228; 276; 394; 750; 914
6 ATACTA 1 8 1 8
7 ATAAAA 5 101; 224; 277; 575; 811 5 101; 224; 277; 575; 811
8 ATGAAA 5 23; 671; 769; 806; 854 5 23; 671; 769; 806; 854
9 AAGCAT 0 NA 0 NA
10 ATTAAT 1 522 1 522
11 ATACAT 1 734 1 734
12 AAAATA 7 226; 578; 618; 838; 7 226; 578; 618; 838;
862; 873; 1137 862; 873; 1137
13 ATTAAA 4 462; 589; 834; 1131 4 462; 589; 834; 1131
14 AATTAA 5 461; 521; 588; 833; 5 461; 521; 588; 833;
1130 1130
15 AATACA 3 261; 303; 733 3 261; 303; 733
16 CATAAA 0 NA 0 NA
Total 45 45
*NA = Not Applicable
TABLE-US-00010
TABLE 10
Table 2 sequences found in the native Cry35Ab1 coding region
(SEQ ID NO: 13) and in the redesigned version (SEQ ID NO: 17)
No. Sites in nt Location in No. Sites in nt Location
native Cry35Ab1 native Cry35Ab1 redesigned in redesigned
Table 2 sequence sequence Cry35Ab1 sequence Cry35Ab1 sequence
Sequence (SEQ ID NO: 13) (SEQ ID NO: 13) (SEQ ID NO: 17) (SEQ ID NO: 17)
1 ATATAT 1 168 0 NA*
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 1 959 0 NA
6 TATTTT 2 609; 1144 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 1 1145 0 NA
9 TTATTT 3 63; 145; 1143 1 1143
10 TTTATT 2 144; 1056 0 NA
11 TAATAA 2 12; 216 1 12
12 ATTTAT 0 NA 0 NA
13 TATATT 2 169; 607 0 NA
14 TTTTAT 1 143 0 NA
15 ATATTT 1 608 0 NA
16 TATTAT 4 171; 549; 604; 1141 1 1141
17 TGTTTG 0 NA 0 NA
18 TTATAT 2 606; 958 0 NA
19 TGTAAT 1 300 0 NA
20 AAATAA 8 26; 192; 227; 275; 2 809; 863
384; 809; 863; 1097
Total 31 5
*NA = Not Applicable
Example 4
Synthetic Coding Region Encoding Bacillus thuringiensis Cry1Ab Core Toxin
[0138] Comparative Sequences. The native DNA sequence encoding Cry1Ab core
toxin is given in SEQ ID NO: 19. This sequence was analyzed to determine
which sequences identified in Table 1 are present in SEQ ID NO: 19 and
their locations. The amino acid sequence encoded by SEQ ID NO:19 was then
reverse translated using the target codon frequencies given in the column
of Table 4 for synthetic genes to be used in maize. The resulting DNA
sequence was analyzed and codons were changed where necessary to remove
unwanted open reading frames and remove unwanted restriction enzyme
recognition sites, while maintaining all sequences identified in Table 1.
The amino acid sequence encoded by SEQ ID NO:19 was preserved. The
resulting DNA sequence is given in SEQ ID NO:21.
[0139] SEQ ID NO:21 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the same number of sequences identified in Table 1. The
resulting sequence, which embodies the present invention, is given in SEQ
ID NO:23. Table 11 shows that the number and location of polyadenylation
signal sequences identified in Table 1 are maintained in SEQ ID NO:23.
Table 12 shows that the number of polyadenylation signal sequences
identified in Tables 2 and 3 are reduced in SEQ ID NO:23 compared to SEQ
ID NO:19.
[0140] The synthetic coding region of SEQ ID NO:23 was optimized for
expression in maize.
[0141] A construct for use in expressing the synthetic coding region of
SEQ ID NO:23 was made by combining the synthetic coding region of SEQ ID
NO:23 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription termination and polyadenylation sequence.
[0142] In one embodiment of such a construct, production of the primary
mRNA transcript comprising SEQ ID NO:23 was driven by a copy of a maize
ubiquitin1 promoter with its native intron1 (U.S. Pat. No. 5,510,474). A
fragment comprising a 3' untranslated region from a maize peroxidase 5
gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984) was used to terminate
transcription. A binary plant transformation plasmid, pDAB1 11449,
containing the aforementioned gene expression cassette, was constructed
and utilized in the production of transgenic maize plants. Plasmid pDAB1
11449 further comprises a herbicide resistance gene comprising a coding
region for aryloxyalknoate dioxygenase (AAD-1 v3; U.S. Pat. No.
7,838,733(82), and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A.
107:20240-5) under the transcriptional control of a sugarcane bacilliform
badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol.
39:1221-30). A fragment comprising a 3' untranslated region from a maize
lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) was used to terminate
transcription.
TABLE-US-00011
TABLE 11
Table 1 sequences found in the native Cry1Ab core toxin coding region
(SEQ ID NO: 19) and in the redesigned version (SEQ ID NO: 23)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Ab Native Cry1Ab redesigned Cry1Ab redesigned Cry1Ab
Table 1 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 19) (SEQ ID NO: 19) (SEQ ID NO: 23) (SEQ ID NO: 23)
1 AATAAA 0 NA* 0 NA
2 AATAAT 3 960, 1126, 1387 3 960, 1126, 1387
3 AACCAA 2 253, 280 0 253, 280
4 ATATAA 2 185, 1391 0 185, 1391
5 AATCAA 2 688, 1129 3 688, 1129, 1639
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 1 1232 1 1232
9 AAGCAT 0 NA 0 NA
10 ATTAAT 1 1636 1 1636
11 ATACAT 2 1366, 1613 2 1366, 1613
12 AAAATA 0 NA 0 NA
13 ATTAAA 3 249, 704, 785 3 249, 704, 785
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 16 NA 17 NA
*NA = Not Applicable
TABLE-US-00012
TABLE 12
Table 1 sequences found in the native Cry1Ab core toxin coding region
(SEQ ID NO: 19) and in the redesigned version (SEQ ID NO: 23)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Ab Native Cry1Ab redesigned Cry1Ab redesigned Cry1Ab
Table 1 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 19) (SEQ ID NO: 19) (SEQ ID NO: 23) (SEQ ID NO: 23)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 1 42 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 2 1097, 1792 0 NA
6 TATTTT 0 NA 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 2 199, 1649 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 1 470 0 NA
11 TAATAA 2 1340, 1386 0 NA
12 ATTTAT 2 503, 799 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 1 110 0 NA
16 TATTAT 2 937, 940 0 NA
17 TGTTTG 1 530 0 NA
18 TTATAT 2 1096, 1791 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 2 959, 1125 1 959
Total 18 1
*NA = Not Applicable
Examples
Synthetic Coding Region Encoding Bacillus thuringiensis CryICa Core Toxin
[0143] Comparative Sequences. The native DNA sequence encoding the Cry35A
core toxin is given in SEQ ID NO:25. This sequence was analyzed to
determine which sequences identified in Table 1 are present in SEQ ID
NO:25 and their locations. The amino acid sequence encoded by SEQ ID
NO:25 was then reverse translated using the target codon frequencies
given in the column of Table 4 for synthetic genes to be used in maize.
The resulting DNA sequence was analyzed and codons were changed where
necessary to remove unwanted open reading frames, and remove unwanted
restriction enzyme recognition sites, while maintaining all sequences
identified in Table 1. The amino acid sequence encoded by SEQ ID NO:25
was preserved. The resulting DNA sequence is given in SEQ ID NO:27. This
sequence will be synthesized and used for comparison with a synthetic
gene designed in accordance with the invention.
[0144] SEQ ID NO:27 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the same number of sequences identified in Table 1. The
resulting sequence, which embodies the present invention, is given in SEQ
ID NO:29. Table 13 shows that the number and location of polyadenylation
signal sequences identified in Table 1 are maintained in SEQ ID NO:29.
Table 14 shows that the number of polyadenylation signal sequences
identified in Tables 2 and 3 are reduced in SEQ ID NO:29 compared to SEQ
ID NO:25.
[0145] DNA of SEQ ID NO:29 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:25 and SEQ ID NO:27.
[0146] The synthetic gene of SEQ ID NO:29 was optimized for expression in
maize.
[0147] A construct for use in expressing the synthetic gene of SEQ ID
NO:29 is made by combining the synthetic gene of SEQ ID NO:29 with a 5'
non-translated region comprising a promoter that functions in plant cells
and a 3' non-translated region comprising a transcription terminator and
polyadenylation sequence.
TABLE-US-00013
TABLE 13
Table 1 sequences found in the native Cry1Ca core toxin coding region
(SEQ ID NO: 25) and in the redesigned version (SEQ ID NO: 29)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Ca Native Cry1Ca redesigned Cry1Ca redesigned Cry1Ca
Table 1 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 25) (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 29)
1 AATAAA 0 NA* 0 NA
2 AATAAT 2 646, 916 2 646, 916
3 AACCAA 0 NA 1 1042
4 ATATAA 2 684, 1757 2 684, 1757
5 AATCAAA 1 1405 1 1405
6 ATACTA 0 NA 0 NA
7 ATAAAA 1 1826 1 1826
8 TGAAA 2 254, 569 2 254, 569
9 AAGCAT 1 335 1 335
10 ATTAAT 7 177, 246, 250, 813, 7 177, 246, 250, 813,
817, 1402, 1534 817, 1402, 1534
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 4 245, 249, 816, 1401 4 245, 249, 816, 1401
14 AATTAA 1 642 1 642
15 AATACA 1 1381 1 1381
16 CATAAA 0 NA 0 NA
Total 22 23
*NA = Not Applicable
TABLE-US-00014
TABLE 14
Table 2 sequences found in the native Cry1Ca core toxin coding region
(SEQ ID NO: 25) and in the redesigned version (SEQ ID NO: 29)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry1Ca Native Cry1Ca redesigned Cry1Ca redesigned Cry1Ca
Table 2 core sequence core sequence core sequence core sequence
Sequence (SEQ ID NO: 25) (SEQ ID NO: 25) (SEQ ID NO: 29) (SEQ ID NO: 29)
1 ATATAT 4 323, 325, 908, 1024 0 NA*
2 TTGTTT NA 0 NA
3 TTTTGT 3 186, 1302, 1512 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 3 324, 1023, 1819 0 NA
6 TATTTT 1 1346 0 NA
7 TTTTTT 1 1326 0 NA
8 ATTTTT 2 529, 959 0 NA
9 TTATTT 1 901 0 NA
10 TTTATT 2 900, 962 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 1 899 0 NA
13 TATATT 2 510, 909 0 NA
14 TTTTAT 2 470, 961 0 NA
15 ATATTT 1 110 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 1 1818 0 NA
19 TGTAAT 1 525 0 NA
20 AAATAA 1 645 1 645
Total 26 1
*NA = Not Applicable
Example 6
Synthetic Coding Region Encoding Bacillus thuringiensis Cry6Aa Toxin
[0148] Comparative Sequences. The native DNA sequence encoding the Cry6Aa
toxin is given in SEQ ID NO:31. This sequence was analyzed to determine
which sequences identified in Table 1 are present in SEQ ID NO:31 and
their locations. The amino acid sequence encoded by SEQ ID NO:31 was then
reverse translated using the target codon frequencies given in the column
of Table 4 for synthetic genes to be used in maize. The resulting DNA
sequence was analyzed and codons were changed where necessary to remove
unwanted open reading frames, and remove unwanted restriction enzyme
recognition sites, while maintaining all sequences identified in Table 1.
The amino acid sequence encoded by SEQ ID NO:31 was preserved. The
resulting DNA sequence is given in SEQ ID NO:33. This sequence will be
synthesized and used for comparison with a synthetic gene designed in
accordance with the invention.
[0149] SEQ ID NO:33 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:35.
Table 15 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:35. Table 16
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:35 compared to SEQ ID NO:31.
[0150] DNA of SEQ ID NO:35 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:31 and SEQ ID NO:33.
[0151] The synthetic coding region of SEQ ID NO:35 was optimized for
expression in maize.
[0152] A construct for use in expressing the synthetic coding region of
SEQ ID NO:35 is made by combining the synthetic coding region of SEQ ID
NO:35 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00015
TABLE 15
Table 1 sequences found in the native Cry6Aa coding region
(SEQ ID NO: 31) and in the redesigned version (SEQ ID NO: 35)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry6Aa Native Cry6Aa redesigned redesigned
Table 1 sequence sequence Cry6Aa sequence Cry6Aa sequence
Sequence (SEQ ID NO: 31) (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 35)
1 AATAAA 1 292 1 292
2 AATAAT 6 430, 1309, 1360, 1384, 6 430, 1309, 1360, 1384,
1402, 1420 1402, 1420
3 AACCAA 0 NA* 0 NA
4 ATATAA 2 824, 1344 2 824, 1344
5 AATCAAA 5 103, 634, 832, 1234, 5 103, 634, 832, 1234,
1270 1270
6 ATACTA 0 NA 0 NA
7 ATAAAA 3 269, 293, 826 3 269, 292, 826
8 ATGAAA 1 794 1 794
9 AAGCAT 0 NA 0 NA
10 ATTAAT 2 919, 1183 2 919, 1183
11 ATACAT 0 NA 1 1275
12 AAAATA 3 530, 806, 1358 3 530, 806, 1358
13 ATTAAA 5 51, 56, 188, 495, 963 5 51, 56, 188, 495, 963
13 AATTAA 7 52, 57, 316, 463, 496, 7 52, 57, 316, 463, 496,
718, 964 718, 964
15 AATACA 2 922, 1238 3 922, 1238, 1274
16 CATAAA 1 664 1 664
Total 38 40
*NA = Not Applicable
TABLE-US-00016
TABLE 16
Table 2 sequences found in the native Cry6Aa coding region
(SEQ ID NO: 31) and in the redesigned version (SEQ ID NO: 35)
No. Sites in nt Location in No. Sites in nt Location in
Native Cry6Aa Native Cry6Aa redesigned redesigned
Table 2 sequence sequence Cry6Aa sequence Cry6Aa sequence
Sequence (SEQ ID NO: 31) (SEQ ID NO: 31) (SEQ ID NO: 35) (SEQ ID NO: 35)
1 ATATAT 4 147, 218, 1275, 1372 0 NA*
2 TTGTTT 1 788 0 NA
3 TTTTGT NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 1 941 0 NA
6 TATTTT 2 388, 489 0 NA
7 TTTTTT NA 0 NA
8 ATTTTT 2 236, 555 0 NA
9 TTATTT 1 113 0 NA
10 TTTATT 1 109, 257 0 NA
11 TAATAA 5 66, 249, 1383, 1401, 1419 0 NA
12 ATTTAT 3 108, 299, 938 0 NA
13 TATATT 2 148, 1373 0 NA
14 TTTTAT 2 1314, 1365 0 NA
15 ATATTT 1 387 0 NA
16 TATTAT 1 111 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 4 247, 301, 940, 1190 0 NA
19 TGTAAT 1 1204 0 NA
20 AAATAA 2 1308, 1359 1 1359
Total 33 1
*NA = Not Applicable
Example 7
Synthetic Coding Region Encoding Sphingobiurn herbicidovorans AAD1
[0153] Comparative Sequences. The native DNA sequence encoding the AAD1
protein is given in SEQ ID NO:37. This sequence was analyzed to determine
which sequences identified in Table 1 are present in SEQ ID NO:37 and
their locations. The amino acid sequence encoded by SEQ ID NO:37 was then
reverse translated using the target codon frequencies given in the column
of Table 4 for synthetic genes to be used in maize. The resulting DNA
sequence was analyzed and codons were changed where necessary to remove
unwanted open reading frames, and remove unwanted restriction enzyme
recognition sites, while maintaining all sequences identified in Table 1.
The amino acid sequence encoded by SEQ ID NO:37 was preserved. The
resulting DNA sequence is given in SEQ ID NO:39. This sequence will be
synthesized and used for comparison with a synthetic gene designed in
accordance with the invention.
[0154] SEQ ID NO:39 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:41.
Table 17 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:41. Table 18
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:41 compared to SEQ ID NO:37.
[0155] DNA of SEQ ID NO:41 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:37 and SEQ ID NO:39.
[0156] The synthetic coding region of SEQ ID NO:41 was optimized for
expression in maize.
[0157] A construct for use in expressing the synthetic coding region of
SEQ ID NO:41 is made by combining the synthetic coding region of SEQ ID
NO:41 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00017
TABLE 17
Table 1 sequences found in the native AADI coding region
(SEQ ID NO: 37) and in the redesigned version (SEQ ID NO: 41)
No. Sites in nt Location No. Sites in nt Location in
Native AAD1 in Native redesigned redesigned
Table 1 sequence AAD1 sequence AAD1 sequence AAD1 sequence
Sequence (SEQ ID NO: 37) (SEQ ID NO: 37) (SEQ ID NO: 41) (SEQ ID NO: 41)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 0 NA 1 652
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 0 NA 0 NA
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 0 NA 0 NA
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 0 1
*NA = Not Applicable
TABLE-US-00018
TABLE 2
Table 2 sequences found in the native AADI coding region
(SEQ ID NO: 37) and in the redesigned version (SEQ ID NO: 41)
No. Sites in nt Location No. Sites in nt Location in
Native AAD1 in Native redesigned redesigned
Table 2 sequence AAD1 sequence AAD1 sequence AAD1 sequence
Sequence (SEQ ID NO: 37) (SEQ ID NO: 37) (SEQ ID NO: 41) (SEQ ID NO: 41)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 1 166 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 0 NA 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 1 0
*NA = Not Applicable
Example 8
Synthetic Coding Region Encoding Aspergillus nidulans Delta-9 Desaturase
[0158] Comparative Sequences. The native DNA sequence encoding the
Aspergillus nidulans Delta-9 Desaturase protein is given in SEQ ID NO:43.
This sequence was analyzed to determine which sequences identified in
Table 1 are present in SEQ ID NO:43 and their locations. The amino acid
sequence encoded by SEQ ID NO:43 was then reverse translated using the
target codon frequencies given in the column of Table 4 for synthetic
genes to be used in maize. The resulting DNA sequence was analyzed and
codons were changed where necessary to remove unwanted open reading
frames and remove unwanted restriction enzyme recognition sites, while
maintaining all sequences identified in Table 1. The amino acid sequence
encoded by SEQ ID NO:43 was preserved. The resulting DNA sequence is
given in SEQ ID NO:45. This sequence will be synthesized and used for
comparison with a synthetic gene designed in accordance with the
invention.
[0159] SEQ ID NO:45 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:47.
Table 1 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:47. Table 20
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:47 compared to SEQ ID NO:43.
[0160] DNA of SEQ ID NO:47 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:43 and SEQ ID NO:45.
[0161] The synthetic coding region of SEQ ID NO:47 was optimized for
expression in maize.
[0162] A construct for use in expressing the synthetic coding region of
SEQ ID NO:47 is made by combining the synthetic coding region of SEQ ID
NO:47 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription termination and polyadenylation sequence.
TABLE-US-00019
TABLE 19
Table 1 sequences found in the native Aspergillus nidulans Delta-9
Desaturase coding region (SEQ ID NO: 43) and in the redesigned version
(SEQ ID NO: 47)
No. Sites in nt Location in No. Sites in nt Location
Native Asp-L19 Native Asp-L19 redesigned in redesigned
Table 1 sequence sequence Asp-L19 sequence Asp-L19 Sequence
Sequence (SEQ ID NO: 43) (SEQ ID NO: 43) (SEQ ID NO: 47) (SEQ ID NO: 47)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 1 1326 1 1326
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 0 NA 0 NA
9 AAGCAT 1 94 1 94
10 ATTAAT 0 NA 0 NA
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 0 NA 0 NA
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 2 2
*NA = Not Applicable
TABLE-US-00020
TABLE 20
Table 2 sequences found in the native Aspergillus nidulans Delta-9
Desaturase coding region (SEQ ID NO: 43) and in the redesigned version
(SEQ ID NO: 47)
No. Sites in nt Location in No. Sites in nt Location
Native Asp-L19 Native Asp-L19 redesigned in redesigned
Table 2 sequence Sequence Asp-L19 Sequence Asp-L19 Sequence
Sequence (SEQ ID NO: 43) (SEQ ID NO: 43) (SEQ ID NO: 47) (SEQ ID NO: 47)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 1 NA 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 1 479 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 1 0
*NA = Not Applicable
Example 9
Synthetic Coding Region Encoding Xerophyta viscosa SAP1
[0163] Comparative Sequences. The native DNA sequence encoding the
Xerophyta viscosa SAP1 protein is given in SEQ ID NO:49. This sequence
was analyzed to determine which sequences identified in Table 1 are
present in SEQ ID NO:49 and their locations. The amino acid sequence
encoded by SEQ ID NO:49 was then reverse translated using the target
codon frequencies given in the column of Table 4 for synthetic genes to
be used in maize. The resulting DNA sequence was analyzed and codons were
changed where necessary to remove unwanted open reading frames and remove
unwanted restriction enzyme recognition sites, while maintaining all
sequences identified in Table 1. The amino acid sequence encoded by SEQ
ID NO:49 was preserved. The resulting DNA sequence is given in SEQ ID
NO:51. This sequence will be synthesized and used for comparison with a
synthetic gene designed in accordance with the invention.
[0164] SEQ ID NO:52 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:53.
Table 1 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:53. Table 21
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:53 compared to SEQ ID NO:49.
[0165] DNA of SEQ ID NO:53 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:49 and SEQ ID NO:51.
[0166] The synthetic coding region of SEQ ID NO:53 was optimized for
expression in maize.
[0167] A construct for use in expressing the synthetic coding region of
SEQ ID NO:53 is made by combining the synthetic coding region of SEQ ID
NO:53 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00021
TABLE 21
Table 1 sequences found in the native Xerophyta viscosa SAPI coding
region (SEQ ID NO: 49) and in the redesigned version (SEQ ID NO: 53)
No. Sites in nt Location in No. Sites in nt Location
Native XvSAP1 Native XvSAP1 redesigned in redesigned
Table 1 sequence sequence XvSAP1 Sequence XvSAP1 sequence
Sequence (SEQ ID NO: 49) (SEQ ID NO: 49) (SEQ ID NO: 53) (SEQ ID NO: 53)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 0 NA 0 NA
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 0 NA 1 25
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 0 NA 0 NA
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 0 1
*NA = Not Applicable
TABLE-US-00022
TABLE 22
Table 2 sequences found in the Native Xerophyta viscosa SAPI coding
region (SEQ ID NO: 49) and in the redesigned version (SEQ ID NO: 53)
No. Sites in nt Location in No. Sites in nt Location
Native XvSAP1 Native XvSAP1 redesigned in redesigned
Table 2 sequence sequence XvSAP1 sequence XvSAP1 sequence
Sequence (SEQ ID NO: 49) (SEQ ID NO: 49) (SEQ ID NO: 53) (SEQ ID NO: 53)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 1 755 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 1 756 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 1 754 0 NA
16 TATTAT 1 665 0 NA
17 TGTTTG 1 696 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 5 0
*NA = Not Applicable
Example 10
Synthetic Coding Region Encoding Aequorea victoria GFP 1
[0168] Comparative Sequences. The native DNA sequence encoding the
Aequorea victoria GFPI is given in SEQ ID NO:55. This sequence was
analyzed to determine which sequences identified in Table 1 are present
in SEQ ID NO:55 and their locations. The amino acid sequence encoded by
SEQ ID NO:55 was then reverse translated using the target codon
frequencies given in the column of Table 4 for synthetic genes to be used
in maize. The resulting DNA sequence was analyzed and codons were changed
where necessary to remove unwanted open reading frames and remove
unwanted restriction enzyme recognition sites, while maintaining all
sequences identified in Table 1. The amino acid sequence encoded by SEQ
ID NO:55 was preserved. The resulting DNA sequence is given in SEQ ID
NO:57. This sequence will be synthesized and used for comparison with a
synthetic gene designed in accordance with the invention.
[0169] SEQ ID NO:57 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:59.
Table 1 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:59. Table 23
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:59 compared to SEQ ID NO:55.
[0170] DNA of SEQ ID NO:59 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:55 and SEQ ID NO:57.
[0171] The synthetic coding region of SEQ ID NO:59 was optimized for
expression in maize.
[0172] A construct for use in expressing the synthetic coding region of
SEQ ID NO:59 is made by combining the synthetic coding region of SEQ ID
NO:59 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00023
TABLE 23
Table 1 sequences found in the native Aequorea victoria GFP1 coding
region (SEQ ID NO: 55) and in the redesigned version (SEQ ID NO: 59)
No. Sites in nt Location in No. Sites in nt Location
Native GFP1 Native GFP1 redesigned in redesigned
Table 1 sequence sequence GFP1 sequence GFP1 sequence
Sequence (SEQ ID NO: 55) (SEQ ID NO: 55) (SEQ ID NO: 59) (SEQ ID NO: 59)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 1 467 1 467
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 1 237 1 237
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 1 450 1 450
12 AAAATA 1 551 1 551
13 ATTAAA 1 511 1 511
14 AATTAA 0 NA 0 NA
15 AATACA 1 425 1 425
16 CATAAA 0 NA 1 480
Total 6 7
*NA = Not Applicable
TABLE-US-00024
TABLE 24
Table 2 sequences found in the native Aequorea victoria GFPI coding
region (SEQ ID NO: 55) and in the redesigned version (SEQ ID NO: 59)
No. Sites in nt Location in No. Sites in nt Location
Native GFP1 Native GFP1 redesigned in redesigned
Table 2 sequence sequence GFP1 sequence GFP1 sequence
Sequence (SEQ ID NO: 55) (SEQ ID NO: 55) (SEQ ID NO: 59) (SEQ ID NO: 59)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 1 293 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 1 137 0 NA
10 TTTATT 1 136 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 1 291 0 NA
14 TTTTAT 1 135 0 NA
15 ATATTT 1 292 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 6 0
*NA = Not Applicable
Example 11
Synthetic Coding Region Encoding Leptosphaeria nodorum FAD9
[0173] Comparative Sequences. The native DNA sequence encoding the
Leptosphaeria nodorum FAD9 protein is given in SEQ ID NO:61. This
sequence was analyzed to determine which sequences identified in Table 1
are present in SEQ ID NO:61 and their locations. The amino acid sequence
encoded by SEQ ID NO:61 was then reverse translated using the target
codon frequencies given in the column of Table 4 for synthetic genes to
be used in maize. The resulting DNA sequence was analyzed and codons were
changed where necessary to remove unwanted open reading frames and remove
unwanted restriction enzyme recognition sites, while maintaining all
sequences identified in Table 1. The amino acid sequence encoded by SEQ
ID NO:61 was preserved. The resulting DNA sequence is given in SEQ ID
NO:63. This sequence will be synthesized and used for comparison with a
synthetic gene designed in accordance with the invention.
[0174] SEQ ID NO:63 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:65.
Table 1 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:65. Table 25
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:65 compared to SEQ ID NO:61.
[0175] DNA of SEQ ID NO:65 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:61 and SEQ ID NO:63.
[0176] The synthetic coding region of SEQ ID NO:65 was optimized for
expression in maize.
[0177] A construct for use in expressing the synthetic coding region of
SEQ ID NO:65 is made by combining the synthetic coding region of SEQ ID
NO:65 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00025
TABLE 25
Table 1 sequences found in the native Leptosphaeria nodorum FAD9 coding
region (SEQ ID NO: 61) and in the redesigned version (SEQ ID NO: 65)
No. Sites in nt Location in No. Sites in nt Location in
Native LnFAD9 Native Ln FAD9 redesigned Ln redesigned Ln
Table 1 sequence sequence FAD9 sequence FAD9 sequence
Sequence (SEQ ID NO: 61) (SEQ ID NO: 61) (SEQ ID NO: 65) (SEQ ID NO: 65)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 0 NA 0 NA
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 0 NA 0 NA
8 ATGAAA 0 NA 0 NA
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 0 NA 0 NA
12 AAAATA 0 NA 0 NA
13 ATTAAA 0 NA 0 NA
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 0 0
*NA = Not Applicable
TABLE-US-00026
TABLE 26
Table 2 sequences found in the native Leptosphaeria nodorum FAD9 coding
region (SEQ ID NO: 61) and in the redesigned version (SEQ ID NO: 65)
No. Sites in nt Location in No. Sites in nt Location in
Native LnFAD9 Native Ln FAD9 redesigned Ln redesigned Ln
Table 2 sequence sequence FAD9 sequence FAD9 sequence
Sequence (SEQ ID NO: 61) (SEQ ID NO: 61) (SEQ ID NO: 65) (SEQ ID NO: 65)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 1 1275 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 0 NA 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 1 NA 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 0 NA 0 NA
16 TATTAT 1 416 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 3 0
*NA = Not Applicable
Example 12
Synthetic Coding Region Encoding Xerophyta viscosa PERJ
[0178] Comparative Sequences. The native DNA sequence encoding the
Xerophyta viscosa PERI protein is given in SEQ ID NO:67. This sequence
was analyzed to determine which sequences identified in Table 1 are
present in SEQ ID NO:67 and their locations. The amino acid sequence
encoded by SEQ ID NO:67 was then reverse translated using the target
codon frequencies given in the column of Table 4 for synthetic genes to
be used in maize. The resulting DNA sequence was analyzed and codons were
changed where necessary to remove unwanted open reading frames and remove
unwanted restriction enzyme recognition sites, while maintaining all
sequences identified in Table 1. The amino acid sequence encoded by SEQ
ID NO:67 was preserved. The resulting DNA sequence is given in SEQ ID
NO:69. This sequence will be synthesized and used for comparison with a
synthetic gene designed in accordance with the invention.
[0179] SEQ ID NO:69 was analyzed and codons were changed to remove
potential polyadenylation signal sequences identified in Table 2, while
maintaining the number of sequences identified in Table 1. The resulting
sequence, which embodies the present invention, is given in SEQ ID NO:71.
Table 1 shows that the number and location of polyadenylation signal
sequences identified in Table 1 are maintained in SEQ ID NO:71. Table 27
shows that the number of polyadenylation signal sequences identified in
Tables 2 and 3 are reduced in SEQ ID NO:71 compared to SEQ ID NO:67.
[0180] DNA of SEQ ID NO:71 is synthesized, and expression levels observed
in plant cells transformed to express this sequence are compared with
expression levels observed in plant cells transformed to express SEQ ID
NO:67 and SEQ ID NO:69.
[0181] The synthetic coding region of SEQ ID NO:71 was optimized for
expression in maize.
[0182] A construct for use in expressing the synthetic coding region of
SEQ ID NO:71 is made by combining the synthetic coding region of SEQ ID
NO:71 with a 5' non-translated region comprising a promoter that
functions in plant cells and a 3' non-translated region comprising a
transcription terminator and polyadenylation sequence.
TABLE-US-00027
TABLE 27
Table 1 sequences found in the native Xerophyta viscosa PERI coding
region (SEQ ID NO: 67) and in the redesigned version (SEQ ID NO: 71)
No. Sites in nt Location in No. Sites in nt Location in
Native XvPER1 Native XvPER1 redesigned redesigned
Table 1 sequence sequence XvPER1 sequence XvPER1 sequence
Sequence (SEQ ID NO: 67) (SEQ ID NO: 67) (SEQ ID NO: 71) (SEQ ID NO: 71)
1 AATAAA 0 NA* 0 NA
2 AATAAT 0 NA 0 NA
3 AACCAA 0 NA 0 NA
4 ATATAA 0 NA 0 NA
5 AATCAA 0 NA 0 NA
6 ATACTA 0 NA 0 NA
7 ATAAAA 1 605 1 605
8 ATGAAA 0 NA 0 NA
9 AAGCAT 0 NA 0 NA
10 ATTAAT 0 NA 0 NA
11 ATACAT 0 NA 0 NA
12 AAAATA 1 282 1 282
13 ATTAAA 0 NA 0 NA
14 AATTAA 0 NA 0 NA
15 AATACA 0 NA 0 NA
16 CATAAA 0 NA 0 NA
Total 2 2
*NA = Not Applicable
TABLE-US-00028
TABLE 28
Table 2 sequences found in the native Xerophyta viscosa PERI coding
region (SEQ ID NO: 67 and in the redesigned version (SEQ ID NO: 71)
No. Sites in nt Location in No. Sites in nt Location in
Native XvPER1 Native XvPER1 redesigned redesigned
Table 1 sequence sequence XvPER1 sequence XvPER1 sequence
Sequence (SEQ ID NO: 67) (SEQ ID NO: 67) (SEQ ID NO: 71) (SEQ ID NO: 71)
1 ATATAT 0 NA* 0 NA
2 TTGTTT 0 NA 0 NA
3 TTTTGT 0 NA 0 NA
4 TGTTTT 0 NA 0 NA
5 TATATA 0 NA 0 NA
6 TATTTT 0 NA 0 NA
7 TTTTTT 0 NA 0 NA
8 ATTTTT 0 NA 0 NA
9 TTATTT 0 NA 0 NA
10 TTTATT 0 NA 0 NA
11 TAATAA 0 NA 0 NA
12 ATTTAT 0 NA 0 NA
13 TATATT 0 NA 0 NA
14 TTTTAT 0 NA 0 NA
15 ATATTT 0 NA 0 NA
16 TATTAT 0 NA 0 NA
17 TGTTTG 0 NA 0 NA
18 TTATAT 0 NA 0 NA
19 TGTAAT 0 NA 0 NA
20 AAATAA 0 NA 0 NA
Total 0 0
*NA = Not Applicable
Example 13
WHISKERS.RTM. Transformation of Maize with Xv SAPI
[0183] A standard WHISKERS transformation vector was constructed in which
the Arabidopsis thaliana promoter. Rd29A, was placed 5' to the XvSAP1
redesigned coding region sequence of the invention (SEQ ID NO:53). These
sequences were flanked by Zea maize PERS, 3' and 5' untranslated regions
to stabilize expression of the redesigned coding region. A pat selection
cassette (See, for example, U.S. Pat. No. 5,648,477) driven by the rice
actin1 promoter was placed 3' to the XvSAP1 expression cassette.
[0184] Vector DNA was digested with appropriate restriction enzymes to
release a fragment containing the bacterial ampicillin resistance gene
present in the vector backbone, and to produce a linear DNA fragment
suitable for WHISKERS.TM.-mediated transformation. Purification of the
linear fragment containing the XvSAP1 and pat expression cassettes was
accomplished on a preparative scale by high pressure liquid
chromatography (HPLC). This plant transformation DNA was delivered into
maize Hi-II suspension cell cultures via WHISKERS.TM.-mediated
transformation (essentially as described in U.S. Pat. Nos. 5,302,523 and
5,464,765; US Patent Publication No. 2008/0182332; and Petolino and
Arnold (2009) (Methods Molec. Biol. 526:59-67).
[0185] Transformants were placed in selective medium after which
transformed isolates were obtained over the course of approximately 8
weeks. The selection medium was an LS based medium (LS Basal medium, N6
vitamins, 1.5 mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic
acid monohydrate; PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM
L-proline, 1.0 mg/L AgNO.sub.3, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum,
pH 5.7) containing Bialaphos (Gold BioTechnology). The embryos were
transferred to selection media containing 3 mg/L Bialaphos until
embryogenic isolates were obtained. Recovered isolates were bulked up by
transferring to fresh selection medium at 2-week intervals for
regeneration and further analysis.
[0186] For regeneration, the cultures were transferred to "28" induction
medium (MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine,
0.25 mg/L 2, 4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan
gum. pH 5.7) for 1 week under low-light conditions (14
.mu.Em-.sup.2s-.sup.1>) then 1 week under high-light conditions
(approximately 89 .mu.Em-.sup.2s-1)_Tissues were subsequently transferred
to "36" regeneration medium (same as induction medium except lacking
plant growth regulators). When plantlets reached 3-5 cm in length, they
were transferred to glass culture tubes containing SHGA medium (Schenk
and Hildebrandt salts and vitamins (1972); PhytoTechnologies Labr.), 1.0
gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/L Gellan gum, pH 5.8) to
allow for further growth and development of the shoot and roots. Plants
were transplanted to the same soil mixture as described earlier herein
and grown to flowering in the greenhouse. Controlled pollinations for
seed production were conducted.
Example 14
Agrobacterium Transformation
[0187] Standard cloning methods are used in the construction of binary
plant transformation and expression plasmids. Restriction endonucleases
and T4 DNA Ligase are obtained from NEB. Plasmid preparations are
performed using the NucleoSpin.RTM. Plasmid Preparation kit or the
NucleoBond.RTM. AX Xtra Midi kit (both from Macherey-Nagel), following
the instructions of the manufacturers. DNA fragments are purified using
the QIAquick.RTM. PCR Purification Kit or the QIAEX II.RTM. Gel
Extraction Kit (both from Qiagen) after gel isolation.
[0188] Synthetic genes in accordance with the invention may be synthesized
by a commercial vendor (e.g. DNA2.0, Menlo Park, Calif.) and supplied as
cloned fragments in standard plasmid vectors, or may be obtained by
standard molecular biology manipulation of other constructs containing
appropriate nucleotide sequences.
[0189] In a non-limiting example, a basic cloning strategy may be to
subclone full length coding sequences (CDS) into a plant expression
plasmid at Neal and Sac! restriction sites. The resulting plant
expression cassettes containing the appropriate coding region under the
control of plant expression elements, (e.g., plant expressible promoters,
3' terminal transcription termination and polyadenylate addition
determinants, and the like) are subcloned into a binary vector plasmid,
utilizing, for example, Gateway.RTM. technology or standard restriction
enzyme fragment cloning procedures. LR Clonase.TM. (Invitrogen) for
example, may be used to recombine the full length and modified gene plant
expression cassettes into a binary plant transformation plasmid if the
Gateway.RTM. technology is utilized. It is convenient to employ a binary
plant transformation vector that harbors a bacterial gene that confers
resistance to the antibiotic spectinomycin when the plasmid is present in
E. coli and Agrobacterium cells. It is also convenient to employ a binary
vector plasmid that contains a plant-expressible selectable marker gene
that is functional in the desired host plants. Examples of
plant-expressible selectable marker genes include but are not limited
those that encode the aminoglycoside phosphotransferase gene (aph!I) of
transposon Tn5, which confers resistance to the antibiotics kanamycin,
neomycin and G418, as well as those genes which code for resistance or
tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin
(bialaphos), imidazolinones, sulfonylureas and triazolopyrimidine
herbicides, such as chlorosulfuron, bromoxynil, dalapon and the like.
[0190] Electro-competent cells of Agrobacterium tumefuciens strain Z707S
(a streptomycin-resistant derivative of Z707; Hepburn et al., 1985, J.
Gen. Microbiol. 131:2961-2969.) are prepared and transformed using
electroporation (Weigel and Glazebrook, 2002, Arabidopsis: A Laboratory
Manual). After electroporation, 1 mL of YEP broth (gm/L: yeast extract,
10; peptone, 10; NaCl, 5) are added to the cuvette and the cell-YEP
suspension is transferred to a 15 mL culture tube for incubation at
28.degree. in a water bath with constant agitation for 4 hours. The cells
are plated on YEP plus agar (25 gm/L) with spectinomycin (200 .mu.g/mL)
and streptomycin (250 .mu.g/mL) and the plates are incubated for 2-4 days
at 280. Well separated single colonies are selected and streaked onto
fresh YEP+agar plates with spectinomycin and streptomycin as before, and
incubated at 28.degree. for 1-3 days.
[0191] The presence of the synthetic gene insert in the binary plant
transformation vector is performed by PCR analysis using vector-specific
primers with template plasmid DNA prepared from selected Agrobacterium
colonies. The cell pellet from a 4 mL aliquot of a 15 mL overnight
culture grown in YEP with spectinomycin and streptomycin as before is
extracted using Qiagen Spin.RTM. Mini Preps, performed per manufacturer's
instructions. Plasmid DNA from the binary vector used in the
Agrobacterium electroporation transformation is included as a control.
The PCR reaction is completed using Taq DNA polymerase from Invitrogen
per manufacture's instructions at 0.5.times. concentrations. PCR
reactions are carried out in a MJ Research Peltier Thermal Cycler
programmed with the following conditions: Step 1) 940 for 3 minutes; Step
2) 940 for 45 seconds; Step 3) 55.degree. for 30 seconds; Step 4)
72.degree. for 1 minute per kb of expected product length; Step 5) 29
times to Step 2; Step 6) 72.degree. for 10 minutes. The reaction is
maintained at 40 after cycling. The amplification products are analyzed
by agarose gel electrophoresis (e.g. 0.7% to 1% agarose, w/v) and
visualized by ethidium bromide staining. A colony is selected whose PCR
product is identical to the plasmid control.
[0192] Alternatively, the plasmid structure of the binary plant
transformation vector containing the synthetic gene insert is performed
by restriction digest fingerprint mapping of plasmid DNA prepared from
candidate Agrobacterium isolates by standard molecular biology methods
well known to those skilled in the art of Agrobacterium manipulation.
[0193] Those skilled in the art of obtaining transformed plants via
Agrobacterium-mediated transformation methods will understand that other
Agrobacterium strains besides Z707S may be used to advantage, and the
choice of strain may depend upon the identity of the host plant species
to be transformed.
Example 15
Production of Insecticidal Proteins in Dicot Plants
[0194] Arabidopsis Transformation. Arabidopsis thaliana Col-01 is
transformed using the floral dip method (Weigel and Glazebrook, supra).
The selected Agrobacterium colony is used to inoculate 1 mL to 15 mL
cultures of YEP broth containing appropriate antibiotics for selection.
The culture is incubated overnight at 28.degree. with constant agitation
at 220 rpm. Each culture is used to inoculate two 500 mL cultures of YEP
broth containing appropriate antibiotics for selection and the new
cultures are incubated overnight at 280 with constant agitation. The
cells are pelleted at approximately 8700.times.g for 10 minutes at room
temperature, and the resulting supernatant is discarded. The cell pellet
is gently resuspended in 500 mL of infiltration media containing:
1/2.times. Murashige and Skoog salts (Sigma-Aldrich)/Gamborg's BS
vitamins (Gold BioTechnology. St. Louis, Mo.), 10% (w/v) sucrose, 0.044
.mu.M benzylaminopurine (10 .mu.L/liter of 1 mg/mL stock in DMSO) and 300
.mu.L/liter Silwet L-77. Plants approximately 1 month old are dipped into
the media for 15 seconds, with care taken to assure submergence of the
newest inflorescence. The plants are then laid on their sides and covered
(transparent or opaque) for 24 hours, washed with water, and placed
upright. The plants are grown at 22.degree., with a 16-hour light/8-hour
dark photoperiod. Approximately 4 weeks after dipping, the seeds are
harvested.
[0195] Arabidopsis Growth and Selection.
[0196] Freshly harvested Ti seed is allowed to dry for at least 7 days at
room temperature in the presence of desiccant. Seed is suspended in a
0.1% agar/water (Sigma-Aldrich) solution and then stratified at 4.degree.
for 2 days. To prepare for planting, Sunshine Mix LP5 (Sun Gro
Horticulture Inc., Bellevue, Wash.) in 10.5 inch.times.21 inch
germination trays (T.O. Plastics Inc., Clearwater, Minn.) is covered with
fine vermiculite, sub-irrigated with Hoagland's solution (Hoagland and
Amon, 1950) until wet, then allowed to drain for 24 hours. Stratified
seed is sown onto the vermiculite and covered with humidity domes (KORD
Products, Bramalea, Ontario, Canada) for 7 days. Seeds are germinated and
plants are grown in a Conviron (Models CMP4030 or CMP3244; Controlled
Environments Limited, Winnipeg, Manitoba, Canada) under long day
conditions (16 hours light/8 hours dark) at a light intensity of 120-150
.mu.moL/nsec under constant temperature (22.degree.) and humidity
(40-50%). Plants are initially watered with Hoagland's solution and
subsequently with deionized water to keep the soil moist but not wet.
[0197] The domes are removed 5-6 days post sowing and plants are sprayed
with a chemical selection agent to kill plants germinated from
nontransformed seeds. For example, if the plant expressible selectable
marker gene provided by the binary plant transformation vector is a pat
or bar gene (Wehrmann et al., 1996, Nat. Biotech. 14:1274-1278),
transformed plants may be selected by spraying with a 1000.times.
solution of Finale (5.78% glufosinate ammonium, Farnam Companies Inc.,
Phoenix, Ariz.). Two subsequent sprays are performed at 5-7 day
intervals. Survivors (plants actively growing) are identified 7-10 days
after the final spraying and transplanted into pots prepared with
Sunshine Mix LP5. Transplanted plants are covered with a humidity dome
for 3-4 days and placed in a Conviron under the above-mentioned growth
conditions.
[0198] Those skilled in the art of dicot plant transformation will
understand that other methods of selection of transformed plants are
available when other plant expressible selectable marker genes (e.g.
herbicide tolerance genes) are used.
[0199] Insect Bioassays of Transgenic Arabidopsis.
[0200] Transgenic Arabidopsis lines expressing Cry proteins are
demonstrated to be active against sensitive insect species in artificial
diet overlay assays. Protein extracted from transgenic and non-transgenic
Arabidopsis lines is quantified by appropriate methods and sample volumes
are adjusted to normalize protein concentration. Bioassays are conducted
on artificial diet as described above. Non-transgenic Arabidopsis and/or
buffer and water are included in assays as background check treatments.
Example 16
Agrobacterium Transformation for Generation of Superbinary Vectors
[0201] The Agrobacterium superbinary system is conveniently used for
transformation of monocot plant hosts. Methodologies for constructing and
validating superbinary vectors are well disclosed and incorporated herein
by reference (Operating Manual for Plasmid pSB 1, Version 3.1, available
from Japan Tobacco, Inc., Tokyo, Japan). Standard molecular biological
and microbiological methods are used to generate superbinary plasmids.
Verification/validation of the structure of the superbinary plasmid is
done using methodologies as described above for binary vectors, and may
be modified as suggested in the Operating Manual for Plasmid pSB 1.
Example 17
Production of Insecticidal Proteins in Monocot Plants
[0202] Agrobacterium-Mediated Transformation of Maize.
[0203] Seeds from a High II F.sub.1 cross (Armstrong et al., 1991, Maize
Genet. Coop. Newsletter 65:92-93) are planted into 5-gallon-pots
containing a mixture of 95% Metro-Mix 360 soilless growing medium (Sun
Gro Horticulture, Bellevue, Wash.) and 5% clay/loam soil. The plants are
grown in a greenhouse using a combination of high pressure sodium and
metal halide lamps with a 16:8 hour Light:Dark photoperiod. For obtaining
immature F.sub.2 embryos for transformation, controlled sib-pollinations
are performed. Immature embryos are isolated at 8-10 days
post-pollination when embryos are approximately 1.0 to 2.0 mm in size.
[0204] Infection and Co-Cultivation.
[0205] Maize ears are surface sterilized by scrubbing with liquid soap,
immersing in 70% ethanol for 2 minutes, and then immersing in 20%
commercial bleach (0.1% sodium hypochlorite) for 30 minutes before being
rinsed with sterile water. A suspension of Agrobacterium cells containing
a superbinary vector is prepared by transferring 1-2 loops of bacteria
grown on YEP solid medium containing 100 mg/L spectinomycin, 10 mg/L
tetracycline, and 250 mg/L streptomycin at 280 for 2-3 days into 5 mL of
liquid infection medium (LS Basal Medium (Linsmaier and Skoog, 1965,
Physiol. Plant. 18:100-127), N6 vitamins (Chu et al., 1975, Scientia
Sinica 18:659-668), 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5
gm/L sucrose, 36.0 gm/L glucose, 6 mM L-proline, pH 5.2) containing 100
.mu.M acetosyringone. The solution was vortexed until a uniform
suspension was achieved, and the concentration is adjusted to a final
density of about 200 Klett units, using a Klett-Summerson colorimeter
with a purple filter, or an optical density of approximately 0.4 at 550
nm. Immature embryos are isolated directly into a micro centrifuge tube
containing 2 mL of the infection medium. The medium is removed and
replaced with 1 mL of the Agrobacterium solution with a density of 200
Klett units, and the Agrobacterium and embryo solution is incubated for 5
minutes at room temperature and then transferred to co-cultivation medium
(LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mM
L-proline, 0.85 mg/L AgNO.sub.3, 100 M acetosyringone, 3.0 gm/L Gellan
gum (PhytoTechnology Laboratories., Lenexa, Kans.), pH 5.8) for 5 days at
250 under dark conditions.
[0206] After co-cultivation, the embryos are transferred to selective
medium after which transformed isolates are obtained over the course of
approximately 8 weeks. For selection of maize tissues transformed with a
superbinary plasmid containing a plant expressible pat or bar selectable
marker gene, an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L
2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate;
PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/L
AgNO.sub.3. 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) is used
with Bialaphos (Gold BioTechnology). The embryos are transferred to
selection media containing 3 mg/L Bialaphos until embryogenic isolates
were obtained. Recovered isolates are bulked up by transferring to fresh
selection medium at 2-week intervals for regeneration and further
analysis.
[0207] Those skilled in the art of maize transformation will understand
that other methods of selection of transformed plants are available when
other plant expressible selectable marker genes (e.g. herbicide tolerance
genes) are used.
[0208] Regeneration and Seed Production.
[0209] For regeneration, the cultures are transferred to "28" induction
medium (MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine,
0.25 mg/L 2, 4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan
gum, pH 5.7) for 1 week under low-light conditions (14
.mu.Em-.sup.2s-.sup.1) then 1 week under high-light conditions
(approximately 89 .mu.Em-.sup.2s-1) Tissues are subsequently transferred
to "36" regeneration medium (same as induction medium except lacking
plant growth regulators). When plantlets grow to 3-5 cm in length, they
were transferred to glass culture tubes containing SHGA medium (Schenk
and Hildebrandt salts and vitamins (1972); PhytoTechnologies Labr.), 1.0
gm/L myo-inositol, 10 gm/L sucrose and 2.0 gm/L Gellan gum. pH 5.8) to
allow for further growth and development of the shoot and roots. Plants
are transplanted to the same soil mixture as described earlier herein and
grown to flowering in the greenhouse. Controlled pollinations for seed
production are conducted.
[0210] Alternatively, binary vectors may be used to produce transgenic
maize plants that contain one or more chimeric genes stably-integrated
into the plant genome and comprising a coding region disclosed herein.
For example, plants comprising at least one coding region of SEQ ID
NOs:5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, or 71 are produced
following Agrobacterium-mediated transformation. Maize transformation
methods employing binary transformation vectors are known in the art. In
one embodiment, transformed tissues are selected by their ability to grow
on haloxyfop-containing medium and are screened for protein production,
as appropriate.
[0211] Ear sterilization and embryo isolation. Maize immature embryos were
obtained from plants of Zea mays inbred line B104 grown in the greenhouse
and self or sib-pollinated to produce ears. The ears were harvested
approximately 9 to 12 days post-pollination. On the experimental day,
de-husked ears were surface-sterilized by immersion in a 20% solution of
sodium hypochlorite (6.15%) and shaken for 20 to 30 min, followed by
three rinses in sterile water. After sterilization, immature zygotic
embryos (1.5 to 2.4 mm) were aseptically dissected from each ear and
randomly distributed into microcentrifuge tubes containing liquid
Inoculation Medium. Inoculation Medium contains: 2.2 gm/L MS salts (Frame
et al., 2011, Genetic Transformation Using Maize Immature Zygotic
Embryos. IN Plant Embryo Culture Methods and Protocols: Methods in
Molecular Biology. T. A. Thorpe and E. C. Yeung, (Eds), SPRINGER SCIENCE
AND BUSINESS MEDIA, LLC. pp 327-341); IX ISU Modified MS Vitamins (Frame
et al, 2011 supra); 68.4 gm/L sucrose; 36 gm/L glucose; 115 mg/L
L-proline; 100 mg/L myo-inositol; and 200 M acetosyringone (prepared in
DMSO); at pH 5.4. For a given set of experiments, embryos from pooled
ears were used for each transformation.
[0212] Agrobacterium Culture Initiation.
[0213] Glycerol stocks of Agrobacterium strain DAt13192 (International PCT
Publication No. WO2012016222(A2)) containing the binary transformation
vector pDAB111440 (Example 1) were streaked on AB minimal medium plates
(Watson, et al., (1975) J. Bacteriol. 123:255-264) containing appropriate
antibiotics and were grown at 20.degree. C. for 3 to 4 days. A single
colony was picked and streaked onto YEP plates (gm/L: yeast extract, 10;
Peptone, 10; NaCl 5) containing the same antibiotics and was incubated at
20.degree. C. for 1-2 days.
[0214] Agrobacterium Culture and Co-Cultivation.
[0215] Agrobacterium colonies were taken from a YEP plate, suspended in 10
mL of Inoculation Medium in a 50 mL disposable tube, and the cell density
was adjusted to an OO.sub.550 of 0.2 to 0.4 (Optical Density measured at
550 nm; a measure of cell growth) using a spectrophotometer. The
Agrobacterium cultures were incubated on a rotary shaker at 125 rpm (room
temperature) while embryo dissection was performed. Immature zygotic
embryos (previously isolated from the sterilized maize kernels and placed
in 1 mL of lnoculation Medium) were washed once in the same medium. Two
ml of the Agrobacterium suspension was added to each tube of embryos and
the tubes were placed on a shaker platform for 10 to 15 minutes. The
embryos were transferred onto Co-cultivation Medium, oriented with the
scutellum facing up, and incubated at 25.degree. C., under 24-hour light
at 50 .mu.Em-.sup.2 sec''; light intensity for 3 days. Co-cultivation
Medium, contains 4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/l
sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH
(3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid); 100
mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L
AgNO.sub.3; 100 .mu.M acetosyringone in DMSO; and 3 gm/v GELZAN.TM.
(SIGMA-ALDRICH); at pH 5.8.
[0216] Callus Selection and Regeneration of Putative Events. Following the
co-cultivation period, embryos were transferred to Resting Medium and
incubated under 24-hour light at 50 .mu.Em-.sup.2 sec-.sup.1 light
intensity and at 25.degree. C. for 3 days. Resting Medium contains 4.33
gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L
L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L
Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 0.5 gm/L MES
(2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES
LABR.; Lenexa, Kans.); 250 mg/L Carbenicillin; and 2.3 gm/L GELZAN.TM.;
at pH 5.8. Embryos were transferred onto Selection Medium 1 (which
consists of the Resting Medium (above) with 100 nM R-Haloxyfop acid
(0.0362 mg/L)), and incubated in either dark and/or under 24-hour light
at 50 .mu.Em-2 sec-.sup.1 light intensity for 7 to 14 days at 28.degree.
C. Proliferating embryogenic calli were transferred onto Selection Medium
2 (which consists of Resting Medium (above), with 500 nM R-Haloxyfop acid
(0.1810 mg/L)), and were incubated in 24-hour light at 50
.mu.Em-.sup.2sec-.sup.1 light intensity for 14 to 21 days at 28.degree.
C. This selection step allowed transgenic callus to further proliferate
and differentiate.
[0217] Proliferating, embryogenic calli were transferred onto
PreRegeneration Medium and cultured under 24-hour light at 50
.mu.Em-.sup.2 sec-.sup.1 light intensity for 7 days at 28.degree. C.
PreRegeneration Medium contains 4.33 gm/L MS salts; IX ISU Modified MS
Vitamins; 45 gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50
mg/L Casein Enzymatic Hydrolysate; 1.0 mg/L AgNO.sub.3; 0.25 gm/L MES;
0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in
ethanol; 1 mg/L 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L
GELZAN.TM.; and 500 nM R-Haloxyfop acid; at pH 5.8. Embryogenic calli
with shoot-like buds were transferred onto Regeneration Medium and
cultured under 24-hour light at 50 .mu.Em-.sup.2 sec'' light intensity
for 7 days. Regeneration Medium I contains 4.33 gm/L MS salts; IX ISU
Modified MS Vitamins; 60 gm/L sucrose; 100 mg/L myo-inositol; 125 mg/L
Carbenicillin; 3.0 gm/L GELZAN.TM.; and 500 nM R-Haloxyfop acid; at pH
5.8. Small shoots with primary roots were transferred to Shoot/Root
medium in PHYTATRAYS (PHYTOTECHNOLOGIES LABR; Lenexa, Kans.) and were
incubated under 16:8 hr. light:dark at 140 to 190 .mu.Em-.sup.2 sec''
light intensity for 7 days at 27.degree. C. Shoot/Root Medium contains
4.33 gm/L MS salts; IX ISU Modified MS Vitamins; 30 gm/L sucrose; 100
mg/L myo-inositol; 3.5 gm/L GELZAN.TM.; at pH 5.8. Putative transgenic
plantlets were analyzed for transgene copy number by quantitative
real-time PCR or other standard molecular analysis techniques, and were
transferred to soil.
[0218] Transfer and establishment of T.sub.0 plants in the greenhouse for
seed production. Transformed plant tissues selected by their ability to
grow on medium containing 500 nM R-Haloxyfop acid were transplanted into
METRO-MIX 360 soilless growing medium (SUN GRO HORTICULTURE) and
hardened-off in a growth room. Plants were then transplanted into
SUNSHINE CUSTOM BLEND 160 soil mixture and grown to flowering in the
greenhouse. Controlled pollinations for seed production are conducted.
[0219] Leaf tissues of selected T.sub.0 plants were sampled at the V-3 to
V-5 stage. Two 6 mm diameter leaf samples were stored in a 96 well
cluster tube rack at -80.degree. C. until the day of analysis. Two
DAISY.TM. steel BB's and 200 .mu.L of extraction buffer (PBS solution
containing 0.05% of Tween 20 and 5 .mu.L/ml of SIGMA protease inhibitor
cocktail (catalog number 9599)) were added to each tube. The samples were
milled in a KLECO bead mill (Visalia, Calif.) for 3 minutes, on maximum
setting. Samples were centrifuged at 3,000.times.g for 5 minutes, then
100 .mu.L of the supernatant were transferred to an empty sample tube.
Another 100 .mu.L of extraction buffer was added to the plant sample and
bead-milled an 3 additional minutes. After centrifuging again, 100 .mu.L
of this extract was combined with the first 100 .mu.L. The combined
supernatants were mixed and analyzed on the same day as the extraction.
[0220] Proteins extracted from measured areas of leaf tissue were analyzed
for expression of CryIFa protein and AAD-1 protein by standard ELISA
(Enzyme-Linked Immunosorbant Assay) or protein immunoblots (western
blots). For CryIFa ELSA detection, reagents from an ENVIROLOGIX ELISA kit
(Cat. No. AP 016 NW VIO; Portland, Me.) were used according to the
manufacturer's instructions. AAD-1 detection was performed by standard
ELISA methodologies (for example, as taught in Ausubel et al. (1995 and
updates) Current Protocols in Molecular Biology, John Wiley and Sons, New
York) using rabbit antibodies prepared against purified AAD-1 protein.
[0221] The ELISA results obtained from extracts of pDAB1 11440-transformed
plants are disclosed in Table 29. Protein levels are expressed as ng of
the subject protein detected per square centimeter of leaf area
harvested.
TABLE-US-00029
TABLE 29
Expression levels ofCry1Fa and AAD-1 proteins extracted from maize
plants transformed with plasmid pDAB1 11440, as detectedby
ELISA methods.
Sample ID Cry1Fa n2/cm.sup.2 AAD-1 n2/cm.sup.2
111440[3]-001.001 2.30 14.0
111440[3]-015.001 3.80 0.0
111440[3]-023.001 3.80 320.0
111440[3]-020.001 5.40 190.0
111440[3]-011.001 17.00 0.0
[0222] Protein extracts of the five pDAB1 11440-transformed plants listed
in Table 29 (as well as extract from a non-transformed negative control
plant) were prepared as above and probed with Cry1Fa antibody on
immunoblots (western blots). Immunoblot procedures were essentially as
described by Gallagher et al. (2008; Immunoblotting and Immunodetection.
Current Protocols in Immunology 8.10.1-8.10.28). Protein samples (80
.mu.L) were mixed with 20 .mu.L of INVITROGEN NuPAGE LDS Sample Buffer,
heated at >90.degree. C. for five min, loaded on an INVITROGEN NuPAGE
4-12% Bis-Tris gel, and run in MOPS SDS Running Buffer (200 Volts for 45
minutes). BIORAD PRECISION PLUS Dual Color Standards were loaded in a
separate lane. Proteins were transferred to 0.2 .mu.M nitrocellulose
membrane by means of an INVITROGEN iBLOT Gel Transfer system according to
the manufacturer's instructions. The membrane was blocked with INVITROGEN
WESTERN BREEZE BLOCKING MIX, then reacted with Primary antibody
(anti-Cry1F Purified Rabbit Antibody No. D0609RA07-A0; Strategic
Diagnostics Inc., Newark, Del.), followed by Secondary antibody
(INVITROGEN Biotinylated goat anti-rabbit antibody.) This was followed by
INVITROGEN HRP-Streptavidin conjugate and reacted bands were detected by
PIERCE SUPERSIGNAL WEST PICO LUMINOL ENHANCER AND STABLE PEROXIDE (No.
34080).
[0223] Positive control lanes contained 0.5 ng or 1.0 ng of purified
Cry1Fa core toxin protein produced by expression of a full length Cry1Fa
coding region in a Pseudomonas jluorescens expression system (See, for
example, US Patent Application No. 20100269223A1). The full-length Cry1Fa
protein was trypsin treated to release the Cry1Fa core toxin segment of
calculated molecular size 68 kDa, which was used as the positive control
standard on the immunoblot. No antibody-reacting bands were detected in
the extract from the negative control plant, while all five transgenic
plant extracts contained a single predominant band (roughly equal in
intensity to the control Cry1Fa proteins) of estimated size somewhat
larger than 75 kDa.
[0224] Methods of Controlling Insect Pests.
[0225] When an insect comes into contact with an effective amount of toxin
delivered via transgenic plant expression the results are typically death
of the insect, or the insects do not feed upon the source which makes the
toxins available to the insects.
Sequence CWU
1
1
7211818DNABacillus thuringiensisCDS(1)..(1818)Native DNA sequence encoding
Bacillus thuringiensis Cry1Fa core toxin 1atg gag aat aat att caa
aat caa tgc gta cct tac aat tgt tta aat 48Met Glu Asn Asn Ile Gln
Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5
10 15 aat cct gaa gta gaa ata tta aat
gaa gaa aga agt act ggc aga tta 96Asn Pro Glu Val Glu Ile Leu Asn
Glu Glu Arg Ser Thr Gly Arg Leu 20 25
30 ccg tta gat ata tcc tta tcg ctt aca cgt
ttc ctt ttg agt gaa ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg
Phe Leu Leu Ser Glu Phe 35 40
45 gtt cca ggt gtg gga gtt gcg ttt gga tta ttt gat
tta ata tgg ggt 192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp
Leu Ile Trp Gly 50 55 60
ttt ata act cct tct gat tgg agc tta ttt ctt tta cag att
gaa caa 240Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile
Glu Gln 65 70 75
80 ttg att gag caa aga ata gaa aca ttg gaa agg aac cgg gca att act
288Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95
aca tta cga ggg tta gca gat agc tat gaa att tat att gaa gca cta
336Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110 aga
gag tgg gaa gca aat cct aat aat gca caa tta agg gaa gat gtg 384Arg
Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125 cgt att cga
ttt gct aat aca gac gac gct tta ata aca gca ata aat 432Arg Ile Arg
Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130
135 140 aat ttt aca ctt aca
agt ttt gaa atc cct ctt tta tcg gtc tat gtt 480Asn Phe Thr Leu Thr
Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150
155 160 caa gcg gcg aat tta cat tta
tca cta tta aga gac gct gta tcg ttt 528Gln Ala Ala Asn Leu His Leu
Ser Leu Leu Arg Asp Ala Val Ser Phe 165
170 175 ggg cag ggt tgg gga ctg gat ata gct
act gtt aat aat cat tat aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala
Thr Val Asn Asn His Tyr Asn 180 185
190 aga tta ata aat ctt att cat aga tat acg aaa
cat tgt ttg gac aca 624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys
His Cys Leu Asp Thr 195 200
205 tac aat caa gga tta gaa aac tta aga ggt act aat act
cga caa tgg 672Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr
Arg Gln Trp 210 215 220
gca aga ttc aat cag ttt agg aga gat tta aca ctt act gta tta
gat 720Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu
Asp 225 230 235 240
atc gtt gct ctt ttt ccg aac tac gat gtt aga aca tat cca att caa
768Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255 acg
tca tcc caa tta aca agg gaa att tat aca agt tca gta att gag 816Thr
Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270 gat tct cca
gtt tct gct aat ata cct aat ggt ttt aat agg gcg gaa 864Asp Ser Pro
Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275
280 285 ttt gga gtt aga ccg
ccc cat ctt atg gac ttt atg aat tct ttg ttt 912Phe Gly Val Arg Pro
Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300 gta act gca gag act gtt aga
agt caa act gtg tgg gga gga cac tta 960Val Thr Ala Glu Thr Val Arg
Ser Gln Thr Val Trp Gly Gly His Leu 305 310
315 320 gtt agt tca cga aat acg gct ggt aac
cgt ata aat ttc cct agt tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn
Arg Ile Asn Phe Pro Ser Tyr 325 330
335 ggg gtc ttc aat cct ggt ggc gcc att tgg att gca
gat gag gat cca 1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala
Asp Glu Asp Pro 340 345
350 cgt cct ttt tat cgg aca tta tca gat cct gtt ttt gtc cga
gga gga 1104Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg
Gly Gly 355 360 365
ttt ggg aat cct cat tat gta ctg ggg ctt agg gga gta gca ttt caa
1152Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380 caa act
ggt acg aac cac acc cga aca ttt aga aat agt ggg acc ata 1200Gln Thr
Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385
390 395 400 gat tct cta gat gaa
atc cca cct cag gat aat agt ggg gca cct tgg 1248Asp Ser Leu Asp Glu
Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405
410 415 aat gat tat agt cat gta tta aat
cat gtt aca ttt gta cga tgg cca 1296Asn Asp Tyr Ser His Val Leu Asn
His Val Thr Phe Val Arg Trp Pro 420 425
430 ggt gag att tca gga agt gat tca tgg aga gct
cca atg ttt tct tgg 1344Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala
Pro Met Phe Ser Trp 435 440
445 acg cac cgt agt gca acc cct aca aat aca att gat ccg
gag agg att 1392Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro
Glu Arg Ile 450 455 460
act caa ata cca ttg gta aaa gca cat aca ctt cag tca ggt act act
1440Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr
465 470 475 480 gtt
gta aga ggg ccc ggg ttt acg gga gga gat att ctt cga cga aca 1488Val
Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495 agt gga gga cca
ttt gct tat act att gtt aat ata aat ggg caa tta 1536Ser Gly Gly Pro
Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu 500
505 510 ccc caa agg tat cgt gca aga
ata cgc tat gcc tct act aca aat cta 1584Pro Gln Arg Tyr Arg Ala Arg
Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515 520
525 aga att tac gta acg gtt gca ggt gaa cgg
att ttt gct ggt caa ttt 1632Arg Ile Tyr Val Thr Val Ala Gly Glu Arg
Ile Phe Ala Gly Gln Phe 530 535
540 aac aaa aca atg gat acc ggt gac cca tta aca ttc
caa tct ttt agt 1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe
Gln Ser Phe Ser 545 550 555
560 tac gca act att aat aca gct ttt aca ttc cca atg agc cag agt
agt 1728Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser
Ser 565 570 575
ttc aca gta ggt gct gat act ttt agt tca ggg aat gaa gtt tat ata
1776Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590 gac aga
ttt gaa ttg att cca gtt act gca aca ttt gaa tag 1818Asp Arg
Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595
600 605 2605PRTBacillus
thuringiensis 2Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys
Leu Asn 1 5 10 15
Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30 Pro Leu Asp Ile Ser
Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35
40 45 Val Pro Gly Val Gly Val Ala Phe Gly
Leu Phe Asp Leu Ile Trp Gly 50 55
60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln
Ile Glu Gln 65 70 75
80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95 Thr Leu Arg Gly
Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100
105 110 Arg Glu Trp Glu Ala Asn Pro Asn Asn
Ala Gln Leu Arg Glu Asp Val 115 120
125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala
Ile Asn 130 135 140
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145
150 155 160 Gln Ala Ala Asn Leu
His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165
170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr
Val Asn Asn His Tyr Asn 180 185
190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp
Thr 195 200 205 Tyr
Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210
215 220 Ala Arg Phe Asn Gln Phe
Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230
235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg
Thr Tyr Pro Ile Gln 245 250
255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270 Asp Ser
Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275
280 285 Phe Gly Val Arg Pro Pro His
Leu Met Asp Phe Met Asn Ser Leu Phe 290 295
300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp
Gly Gly His Leu 305 310 315
320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335 Gly Val Phe
Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340
345 350 Arg Pro Phe Tyr Arg Thr Leu Ser
Asp Pro Val Phe Val Arg Gly Gly 355 360
365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val
Ala Phe Gln 370 375 380
Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385
390 395 400 Asp Ser Leu Asp
Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405
410 415 Asn Asp Tyr Ser His Val Leu Asn His
Val Thr Phe Val Arg Trp Pro 420 425
430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe
Ser Trp 435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450
455 460 Thr Gln Ile Pro Leu
Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470
475 480 Val Val Arg Gly Pro Gly Phe Thr Gly Gly
Asp Ile Leu Arg Arg Thr 485 490
495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln
Leu 500 505 510 Pro
Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515
520 525 Arg Ile Tyr Val Thr Val
Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535
540 Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr
Phe Gln Ser Phe Ser 545 550 555
560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575 Phe Thr
Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580
585 590 Asp Arg Phe Glu Leu Ile Pro
Val Thr Ala Thr Phe Glu 595 600
605 31818DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus
thuringiensis Cry1Fa core toxin using codons optimized for maize
and Table 1 sequences are maintainedCDS(1)..(1818) 3atg gag aat aat atc
cag aat caa tgc gtg cct tac aat tgt tta aat 48Met Glu Asn Asn Ile
Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5
10 15 aat ccc gag gtg gag ata tta
aac gag gag aga tcc act ggc aga ctg 96Asn Pro Glu Val Glu Ile Leu
Asn Glu Glu Arg Ser Thr Gly Arg Leu 20
25 30 cca ctc gat ata tcc ttg tcc ctt acc
cgt ttc ctt ttg agc gaa ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr
Arg Phe Leu Leu Ser Glu Phe 35 40
45 gtt cct ggt gtg gga gtg gct ttc gga tta ttt
gat ctg ata tgg ggt 192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe
Asp Leu Ile Trp Gly 50 55 60
ttt atc act cct tct gat tgg agc tta ttt ctt ctc cag
att gag caa 240Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln
Ile Glu Gln 65 70 75
80 ttg att gag cag aga ata gaa acc ttg gaa agg aac cgt gca atc
acg 288Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile
Thr 85 90 95
acc ttg cgc ggt ctc gcc gat agc tat gaa att tat att gaa gca ctg
336Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110 agg
gag tgg gag gcc aac cct aat aat gct caa tta agg gaa gat gtg 384Arg
Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val
115 120 125 cgt att cgt
ttt gct aat aca gac gac gct tta ata aca gca ata aat 432Arg Ile Arg
Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn 130
135 140 aat ttc aca ctt aca
tcc ttt gaa atc ccg ctt tta tca gtg tac gtt 480Asn Phe Thr Leu Thr
Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145 150
155 160 caa gcc gcc aat ctc cat tta
tca ctt ctg agg gac gct gtc tcc ttt 528Gln Ala Ala Asn Leu His Leu
Ser Leu Leu Arg Asp Ala Val Ser Phe 165
170 175 ggg caa ggt tgg gga ctg gat atc gct
act gtt aat aat cac tac aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala
Thr Val Asn Asn His Tyr Asn 180 185
190 aga tta ata aac ctg att cat aga tat acg aag
cat tgt ttg gac aca 624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys
His Cys Leu Asp Thr 195 200
205 tac aat caa gga ctg gag aac ctt agg gga act aac act
agg cag tgg 672Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr
Arg Gln Trp 210 215 220
gca agg ttc aac cag ttc aga cgt gat ctc aca ctt act gtg ctg
gat 720Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu
Asp 225 230 235 240
atc gtt gct ctc ttt ccg aac tac gat gtt cgc acc tac cca atc cag
768Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln
245 250 255 acg
tca tcc caa tta aca agg gaa att tat acc tcc tca gtg att gag 816Thr
Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile Glu
260 265 270 gac tct ccc
gtt tct gct aac ata cct aac ggc ttc aac cgc gcc gag 864Asp Ser Pro
Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275
280 285 ttc gga gtt aga ccg
ccc cac ctt atg gac ttt atg aat agc ttg ttt 912Phe Gly Val Arg Pro
Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300 gtg act gct gag act gtt aga
agc caa act gtg tgg ggc ggc cac ttg 960Val Thr Ala Glu Thr Val Arg
Ser Gln Thr Val Trp Gly Gly His Leu 305 310
315 320 gtc agc tca cgc aac acg gct ggc aac
cgt atc aac ttc ccg tct tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn
Arg Ile Asn Phe Pro Ser Tyr 325 330
335 ggg gtc ttt aac cct ggt ggc gcc att tgg att gca
gac gag gac cca 1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala
Asp Glu Asp Pro 340 345
350 cgt cct ttt tat cgc acc ctg tca gat cct gtt ttt gtc aga
ggc gga 1104Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg
Gly Gly 355 360 365
ttt ggg aat cct cat tat gtc ctg ggc ctt agg gga gtg gct ttc caa
1152Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln
370 375 380 cag
act ggc acc aac cac acc cgt acg ttt cgc aat agc ggg acc ata 1200Gln
Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385
390 395 400 gat tct ctt gat
gaa atc cca cct caa gat aac agc ggc gca cct tgg 1248Asp Ser Leu Asp
Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405
410 415 aac gat tat tcc cac gta tta
aat cac gtt acg ttc gtc cgc tgg ccg 1296Asn Asp Tyr Ser His Val Leu
Asn His Val Thr Phe Val Arg Trp Pro 420
425 430 ggt gag atc agc ggc agc gat tca tgg
aga gca cca atg ttt tct tgg 1344Gly Glu Ile Ser Gly Ser Asp Ser Trp
Arg Ala Pro Met Phe Ser Trp 435 440
445 acg cac cgt tca gcc acc cct aca aat aca att gac
ccg gag agg att 1392Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp
Pro Glu Arg Ile 450 455 460
act caa atc cca ttg gtc aaa gca cat aca ctt cag tct ggg acc
acc 1440Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr
Thr 465 470 475 480
gtg gtc aga ggg cct ggg ttc acg gga gga gac att ctt agg cgc aca
1488Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr
485 490 495 tcc gga
gga ccc ttc gct tat act atc gtt aat ata aat ggg cag ctc 1536Ser Gly
Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu
500 505 510 ccc cag cgc tat
cgt gcc aga atc cgt tac gcc tct act aca aat ctc 1584Pro Gln Arg Tyr
Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu 515
520 525 aga atc tac gtg acg gtt gcc
ggt gag cgc att ttt gct ggt cag ttt 1632Arg Ile Tyr Val Thr Val Ala
Gly Glu Arg Ile Phe Ala Gly Gln Phe 530 535
540 aac aag acg atg gat act ggc gac cca ctg
aca ttc caa tct ttc tca 1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu
Thr Phe Gln Ser Phe Ser 545 550 555
560 tac gca act att aat aca gct ttc aca ttc cca atg agc
cag tca tct 1728Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser
Gln Ser Ser 565 570
575 ttc acc gtc ggt gct gat acc ttc agc tct ggc aac gaa gtt tat
ata 1776Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr
Ile 580 585 590
gac aga ttt gag ttg att cca gtt act gca acg ttt gag tga
1818Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu
595 600 605
4605PRTArtificial SequenceSynthetic Construct 4Met Glu Asn Asn Ile Gln
Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn 1 5
10 15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg
Ser Thr Gly Arg Leu 20 25
30 Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu
Phe 35 40 45 Val
Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly 50
55 60 Phe Ile Thr Pro Ser Asp
Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65 70
75 80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg
Asn Arg Ala Ile Thr 85 90
95 Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu
100 105 110 Arg Glu
Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val 115
120 125 Arg Ile Arg Phe Ala Asn Thr
Asp Asp Ala Leu Ile Thr Ala Ile Asn 130 135
140 Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu
Ser Val Tyr Val 145 150 155
160 Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe
165 170 175 Gly Gln Gly
Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn 180
185 190 Arg Leu Ile Asn Leu Ile His Arg
Tyr Thr Lys His Cys Leu Asp Thr 195 200
205 Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr
Arg Gln Trp 210 215 220
Ala Arg Phe Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225
230 235 240 Ile Val Ala Leu
Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245
250 255 Thr Ser Ser Gln Leu Thr Arg Glu Ile
Tyr Thr Ser Ser Val Ile Glu 260 265
270 Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg
Ala Glu 275 280 285
Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe 290
295 300 Val Thr Ala Glu Thr
Val Arg Ser Gln Thr Val Trp Gly Gly His Leu 305 310
315 320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg
Ile Asn Phe Pro Ser Tyr 325 330
335 Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp
Pro 340 345 350 Arg
Pro Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355
360 365 Phe Gly Asn Pro His Tyr
Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370 375
380 Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg
Asn Ser Gly Thr Ile 385 390 395
400 Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp
405 410 415 Asn Asp
Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro 420
425 430 Gly Glu Ile Ser Gly Ser Asp
Ser Trp Arg Ala Pro Met Phe Ser Trp 435 440
445 Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp
Pro Glu Arg Ile 450 455 460
Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465
470 475 480 Val Val Arg
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485
490 495 Ser Gly Gly Pro Phe Ala Tyr Thr
Ile Val Asn Ile Asn Gly Gln Leu 500 505
510 Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr
Thr Asn Leu 515 520 525
Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530
535 540 Asn Lys Thr Met
Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser 545 550
555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr
Phe Pro Met Ser Gln Ser Ser 565 570
575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val
Tyr Ile 580 585 590
Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595
600 605 51818DNAArtificial SequenceSynthetic DNA
sequence in accordance with the invention encoding Bacillus
thuringiensis Cry1Fa core toxin using codons optimized for maize and
with sequences identified in Table 2 removed and Table 1 sequences
are maintainedCDS(1)..(1818) 5atg gag aat aat atc cag aat caa tgc gtg cct
tac aat tgt ctc aat 48Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro
Tyr Asn Cys Leu Asn 1 5 10
15 aat ccc gag gtg gag ata tta aac gag gag aga tcc act
ggc aga ctg 96Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr
Gly Arg Leu 20 25 30
cca ctc gac ata tcc ttg tcc ctt acc cgt ttc ctt ttg agc gaa
ttt 144Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu
Phe 35 40 45
gtt cct ggt gtg gga gtg gct ttc gga ctg ttc gat ctg ata tgg ggc
192Val Pro Gly Val Gly Val Ala Phe Gly Leu Phe Asp Leu Ile Trp Gly
50 55 60 ttt
atc act cct tct gat tgg agc ctc ttc ctt ctc cag att gag caa 240Phe
Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln 65
70 75 80 ttg att gag
cag aga ata gaa acc ttg gaa agg aac cgt gca atc acg 288Leu Ile Glu
Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95 acc ttg cgc ggt ctc
gcc gat agc tat gaa atc tac att gaa gca ctg 336Thr Leu Arg Gly Leu
Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100
105 110 agg gag tgg gag gcc aac ccc
aat aat gct caa tta agg gaa gat gtg 384Arg Glu Trp Glu Ala Asn Pro
Asn Asn Ala Gln Leu Arg Glu Asp Val 115 120
125 cgt att cgt ttt gct aat aca gac gac
gct ctc atc aca gca atc aat 432Arg Ile Arg Phe Ala Asn Thr Asp Asp
Ala Leu Ile Thr Ala Ile Asn 130 135
140 aat ttc aca ctt aca tcc ttt gaa atc ccg ctt
ttg agc gtg tac gtt 480Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu
Leu Ser Val Tyr Val 145 150 155
160 caa gcc gcc aat ctc cac ctc tca ctt ctg agg gac gct
gtc tcc ttt 528Gln Ala Ala Asn Leu His Leu Ser Leu Leu Arg Asp Ala
Val Ser Phe 165 170
175 ggg caa ggt tgg gga ctg gat atc gct act gtg aat aat cac tac
aat 576Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr
Asn 180 185 190
aga tta atc aac ctg att cat aga tat acg aag cac tgc ttg gac aca
624Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr
195 200 205 tac
aat caa gga ctg gag aac ctt agg gga act aac act agg cag tgg 672Tyr
Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210
215 220 gca agg ttc
aac cag ttc aga cgt gat ctc aca ctt act gtg ctg gat 720Ala Arg Phe
Asn Gln Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225
230 235 240 atc gtt gct ctc ttt
ccg aac tac gat gtt cgc acc tac cca atc cag 768Ile Val Ala Leu Phe
Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln 245
250 255 acg tca tcc caa tta aca agg
gaa atc tac acc tcc tca gtg att gag 816Thr Ser Ser Gln Leu Thr Arg
Glu Ile Tyr Thr Ser Ser Val Ile Glu 260
265 270 gac tct ccc gtt tct gct aac ata cct
aac ggc ttc aac cgc gcc gag 864Asp Ser Pro Val Ser Ala Asn Ile Pro
Asn Gly Phe Asn Arg Ala Glu 275 280
285 ttc gga gtt aga ccg ccc cac ctt atg gac ttt
atg aat agc ttg ttc 912Phe Gly Val Arg Pro Pro His Leu Met Asp Phe
Met Asn Ser Leu Phe 290 295 300
gtg act gct gag act gtt aga agc caa act gtg tgg ggc
ggc cac ttg 960Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly
Gly His Leu 305 310 315
320 gtc agc tca cgc aac acg gct ggc aac cgt atc aac ttc ccg tct
tac 1008Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser
Tyr 325 330 335
ggg gtc ttt aac cct ggt ggc gcc att tgg att gca gac gag gac cca
1056Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro
340 345 350 cgt cct
ttt tac cgc acc ctg tca gat ccg gtt ttc gtc aga ggc gga 1104Arg Pro
Phe Tyr Arg Thr Leu Ser Asp Pro Val Phe Val Arg Gly Gly 355
360 365 ttt ggg aat cct cat
tat gtc ctg ggc ctt agg gga gtg gct ttc caa 1152Phe Gly Asn Pro His
Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln 370
375 380 cag act ggc acc aac cac acc
cgt acg ttt cgc aat agc ggg acc ata 1200Gln Thr Gly Thr Asn His Thr
Arg Thr Phe Arg Asn Ser Gly Thr Ile 385 390
395 400 gat tct ctt gat gaa atc cca cct caa gat
aac agc ggc gca cct tgg 1248Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp
Asn Ser Gly Ala Pro Trp 405 410
415 aac gat tat tcc cac gta tta aat cac gtt acg ttc gtc
cgc tgg ccg 1296Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val
Arg Trp Pro 420 425 430
ggt gag atc agc ggc agc gat tca tgg aga gca cca atg ttc tct tgg
1344Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp
435 440 445 acg
cac cgt tca gcc acc cct aca aat aca att gac ccg gag agg att 1392Thr
His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450
455 460 act caa atc cca
ttg gtc aaa gca cat aca ctt cag tct ggg acc acc 1440Thr Gln Ile Pro
Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465
470 475 480 gtg gtc aga ggg cct ggg
ttc acg gga gga gac att ctt agg cgc aca 1488Val Val Arg Gly Pro Gly
Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr 485
490 495 tcc gga gga ccc ttc gct tat act atc
gtt aat ata aat ggg cag ctc 1536Ser Gly Gly Pro Phe Ala Tyr Thr Ile
Val Asn Ile Asn Gly Gln Leu 500 505
510 ccc cag cgc tat cgt gcc aga atc cgt tac gcc tct
act aca aat ctc 1584Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser
Thr Thr Asn Leu 515 520 525
aga atc tac gtg acg gtt gcc ggt gag cgc atc ttt gct ggt cag
ttt 1632Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln
Phe 530 535 540
aac aag acg atg gat act ggc gac cca ctg aca ttc caa tct ttc tca
1680Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser
545 550 555 560 tac
gca act att aat aca gct ttc aca ttc cca atg agc cag tca tct 1728Tyr
Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser
565 570 575 ttc acc gtc ggt
gct gat acc ttc agc tct ggc aac gaa gtc tat atc 1776Phe Thr Val Gly
Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile 580
585 590 gac aga ttt gag ttg att
cca gtt act gca acg ttt gag tga 1818Asp Arg Phe Glu Leu Ile
Pro Val Thr Ala Thr Phe Glu 595
600 605 6605PRTArtificial
SequenceSynthetic Construct 6Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro
Tyr Asn Cys Leu Asn 1 5 10
15 Asn Pro Glu Val Glu Ile Leu Asn Glu Glu Arg Ser Thr Gly Arg Leu
20 25 30 Pro Leu
Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe 35
40 45 Val Pro Gly Val Gly Val Ala
Phe Gly Leu Phe Asp Leu Ile Trp Gly 50 55
60 Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu
Gln Ile Glu Gln 65 70 75
80 Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr
85 90 95 Thr Leu Arg
Gly Leu Ala Asp Ser Tyr Glu Ile Tyr Ile Glu Ala Leu 100
105 110 Arg Glu Trp Glu Ala Asn Pro Asn
Asn Ala Gln Leu Arg Glu Asp Val 115 120
125 Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr
Ala Ile Asn 130 135 140
Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val 145
150 155 160 Gln Ala Ala Asn
Leu His Leu Ser Leu Leu Arg Asp Ala Val Ser Phe 165
170 175 Gly Gln Gly Trp Gly Leu Asp Ile Ala
Thr Val Asn Asn His Tyr Asn 180 185
190 Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu
Asp Thr 195 200 205
Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp 210
215 220 Ala Arg Phe Asn Gln
Phe Arg Arg Asp Leu Thr Leu Thr Val Leu Asp 225 230
235 240 Ile Val Ala Leu Phe Pro Asn Tyr Asp Val
Arg Thr Tyr Pro Ile Gln 245 250
255 Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Thr Ser Ser Val Ile
Glu 260 265 270 Asp
Ser Pro Val Ser Ala Asn Ile Pro Asn Gly Phe Asn Arg Ala Glu 275
280 285 Phe Gly Val Arg Pro Pro
His Leu Met Asp Phe Met Asn Ser Leu Phe 290 295
300 Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val
Trp Gly Gly His Leu 305 310 315
320 Val Ser Ser Arg Asn Thr Ala Gly Asn Arg Ile Asn Phe Pro Ser Tyr
325 330 335 Gly Val
Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro 340
345 350 Arg Pro Phe Tyr Arg Thr Leu
Ser Asp Pro Val Phe Val Arg Gly Gly 355 360
365 Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly
Val Ala Phe Gln 370 375 380
Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile 385
390 395 400 Asp Ser Leu
Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp 405
410 415 Asn Asp Tyr Ser His Val Leu Asn
His Val Thr Phe Val Arg Trp Pro 420 425
430 Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met
Phe Ser Trp 435 440 445
Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile 450
455 460 Thr Gln Ile Pro
Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr 465 470
475 480 Val Val Arg Gly Pro Gly Phe Thr Gly
Gly Asp Ile Leu Arg Arg Thr 485 490
495 Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val Asn Ile Asn Gly
Gln Leu 500 505 510
Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
515 520 525 Arg Ile Tyr Val
Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe 530
535 540 Asn Lys Thr Met Asp Thr Gly Asp
Pro Leu Thr Phe Gln Ser Phe Ser 545 550
555 560 Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met
Ser Gln Ser Ser 565 570
575 Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile
580 585 590 Asp Arg Phe
Glu Leu Ile Pro Val Thr Ala Thr Phe Glu 595 600
605 7372DNABacillus thuringiensisCDS(1)..(372)Native DNA
sequence encoding Bacillus thuringiensis Cry34Ab1 toxin 7atg tca gca
cgt gaa gta cac att gat gta aat aat aag aca ggt cat 48Met Ser Ala
Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1
5 10 15 aca tta caa tta gaa
gat aaa aca aaa ctt gat ggt ggt aga tgg cga 96Thr Leu Gln Leu Glu
Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20
25 30 aca tca cct aca aat gtt gct
aat gat caa att aaa aca ttt gta gca 144Thr Ser Pro Thr Asn Val Ala
Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40
45 gaa tca aat ggt ttt atg aca ggt aca
gaa ggt act ata tat tat agt 192Glu Ser Asn Gly Phe Met Thr Gly Thr
Glu Gly Thr Ile Tyr Tyr Ser 50 55
60 ata aat gga gaa gca gaa att agt tta tat ttt
gac aat cct ttt gca 240Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe
Asp Asn Pro Phe Ala 65 70 75
80 ggt tct aat aaa tat gat gga cat tcc aat aaa tct caa
tat gaa att 288Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln
Tyr Glu Ile 85 90
95 att acc caa gga gga tca gga aat caa tct cat gtt acg tat act
att 336Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr
Ile 100 105 110
caa acc aca tcc tca cga tat ggg cat aaa tca taa
372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser
115 120
8123PRTBacillus thuringiensis 8Met Ser Ala Arg Glu Val His Ile Asp Val
Asn Asn Lys Thr Gly His 1 5 10
15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp
Arg 20 25 30 Thr
Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35
40 45 Glu Ser Asn Gly Phe Met
Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55
60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe
Asp Asn Pro Phe Ala 65 70 75
80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile
85 90 95 Ile Thr
Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100
105 110 Gln Thr Thr Ser Ser Arg Tyr
Gly His Lys Ser 115 120
9372DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus
thuringiensis Cry34Ab1 toxin using codons optimized for maize and
Table 1 sequences are maintainedCDS(1)..(372) 9atg tca gca cgg gag gtc
cac atc gat gta aat aat aag acg ggt cat 48Met Ser Ala Arg Glu Val
His Ile Asp Val Asn Asn Lys Thr Gly His 1 5
10 15 aca tta cag ttg gag gat aaa aca aag
cta gac ggt ggc aga tgg aga 96Thr Leu Gln Leu Glu Asp Lys Thr Lys
Leu Asp Gly Gly Arg Trp Arg 20 25
30 acc agt ccg acc aac gtt gct aac gat caa att aaa
aca ttt gta gcc 144Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys
Thr Phe Val Ala 35 40 45
gaa tca aac ggt ttt atg act ggc acg gag ggg act ata tat
tat tcc 192Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr
Tyr Ser 50 55 60
atc aac gga gaa gcc gag att tcg tta tat ttt gac aat cca ttc gcg
240Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala
65 70 75 80 ggg
tct aat aaa tac gac gga cac tcc aat aaa tct caa tat gaa atc 288Gly
Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile
85 90 95 att aca caa
ggc ggc agc gga aat caa agc cac gtc acg tat act atc 336Ile Thr Gln
Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100
105 110 cag acc act tca tcg
cgc tac ggg cat aaa tca tag 372Gln Thr Thr Ser Ser
Arg Tyr Gly His Lys Ser 115
120 10123PRTArtificial
SequenceSynthetic Construct 10Met Ser Ala Arg Glu Val His Ile Asp Val Asn
Asn Lys Thr Gly His 1 5 10
15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg
20 25 30 Thr Ser
Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35
40 45 Glu Ser Asn Gly Phe Met Thr
Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55
60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp
Asn Pro Phe Ala 65 70 75
80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile
85 90 95 Ile Thr Gln
Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100
105 110 Gln Thr Thr Ser Ser Arg Tyr Gly
His Lys Ser 115 120
11372DNAArtificial SequenceSynthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry34Ab1 toxin using
codons optimized for maize and with sequences identified in Table 2
removed and Table 1 sequences are maintainedCDS(1)..(372) 11atg tca gca
cgg gag gtc cac atc gat gta aat aat aag acg ggt cat 48Met Ser Ala
Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1
5 10 15 aca tta cag ttg
gag gat aaa aca aag cta gac ggt ggc aga tgg aga 96Thr Leu Gln Leu
Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20
25 30 acc agt ccg acc aac
gtt gct aac gat caa att aaa aca ttt gta gcc 144Thr Ser Pro Thr Asn
Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35
40 45 gaa tca aac ggt ttc atg act
ggc acg gag ggg act atc tac tac tcc 192Glu Ser Asn Gly Phe Met Thr
Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55
60 atc aac gga gaa gcc gag att tcg ctg
tac ttc gac aat cca ttc gcg 240Ile Asn Gly Glu Ala Glu Ile Ser Leu
Tyr Phe Asp Asn Pro Phe Ala 65 70
75 80 ggg tct aat aaa tac gac gga cac tcc aat aaa
tct caa tat gaa atc 288Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys
Ser Gln Tyr Glu Ile 85 90
95 att aca caa ggc ggc agc gga aat caa agc cac gtc acg
tat act atc 336Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr
Tyr Thr Ile 100 105 110
cag acc act tca tcg cgc tac ggg cat aaa tca tag
372Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser
115 120
12123PRTArtificial SequenceSynthetic Construct 12Met Ser Ala Arg Glu
Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5
10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu
Asp Gly Gly Arg Trp Arg 20 25
30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val
Ala 35 40 45 Glu
Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50
55 60 Ile Asn Gly Glu Ala Glu
Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 65 70
75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys
Ser Gln Tyr Glu Ile 85 90
95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile
100 105 110 Gln Thr
Thr Ser Ser Arg Tyr Gly His Lys Ser 115 120
131152DNABacillus thuringiensisCDS(1)..(1152)Native DNA sequence
encoding Bacillus thuringiensis Cry35Ab1 toxin 13atg tta gat act aat
aaa gtt tat gaa ata agc aat cat gct aat gga 48Met Leu Asp Thr Asn
Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5
10 15 cta tat gca gca act tat tta
agt tta gat gat tca ggt gtt agt tta 96Leu Tyr Ala Ala Thr Tyr Leu
Ser Leu Asp Asp Ser Gly Val Ser Leu 20
25 30 atg aat aaa aat gat gat gat att gat
gat tat aac tta aaa tgg ttt 144Met Asn Lys Asn Asp Asp Asp Ile Asp
Asp Tyr Asn Leu Lys Trp Phe 35 40
45 tta ttt cct att gat gat gat caa tat att att
aca agc tat gca gca 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile
Thr Ser Tyr Ala Ala 50 55 60
aat aat tgt aaa gtt tgg aat gtt aat aat gat aaa ata
aat gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile
Asn Val Ser 65 70 75
80 act tat tct tca aca aat tca ata caa aaa tgg caa ata aaa gct
aat 288Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala
Asn 85 90 95
ggt tct tca tat gta ata caa agt gat aat gga aaa gtc tta aca gca
336Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110 gga
acc ggt caa gct ctt gga ttg ata cgt tta act gat gaa tcc tca 384Gly
Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser
115 120 125 aat aat ccc
aat caa caa tgg aat tta act tct gta caa aca att caa 432Asn Asn Pro
Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130
135 140 ctt cca caa aaa cct
ata ata gat aca aaa tta aaa gat tat ccc aaa 480Leu Pro Gln Lys Pro
Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150
155 160 tat tca cca act gga aat ata
gat aat gga aca tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn Ile
Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175 gga tgg aca tta gta cct tgt att atg
gta aat gat cca aat ata gat 576Gly Trp Thr Leu Val Pro Cys Ile Met
Val Asn Asp Pro Asn Ile Asp 180 185
190 aaa aat act caa att aaa act act cca tat tat
att tta aaa aaa tat 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr
Ile Leu Lys Lys Tyr 195 200
205 caa tat tgg caa cga gca gta gga agt aat gta gct tta
cgt cca cat 672Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu
Arg Pro His 210 215 220
gaa aaa aaa tca tat act tat gaa tgg ggc aca gaa ata gat caa
aaa 720Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln
Lys 225 230 235 240
aca aca att ata aat aca tta gga ttt caa atc aat ata gat tca gga
768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly
245 250 255 atg
aaa ttt gat ata cca gaa gta ggt gga ggt aca gat gaa ata aaa 816Met
Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys
260 265 270 aca caa cta
aat gaa gaa tta aaa ata gaa tat agt cat gaa act aaa 864Thr Gln Leu
Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275
280 285 ata atg gaa aaa tat
caa gaa caa tct gaa ata gat aat cca act gat 912Ile Met Glu Lys Tyr
Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300 caa tca atg aat tct ata gga
ttt ctt act att act tcc tta gaa tta 960Gln Ser Met Asn Ser Ile Gly
Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310
315 320 tat aga tat aat ggc tca gaa att cgt
ata atg caa att caa acc tca 1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg
Ile Met Gln Ile Gln Thr Ser 325 330
335 gat aat gat act tat aat gtt act tct tat cca aat
cat caa caa gct 1056Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn
His Gln Gln Ala 340 345
350 tta tta ctt ctt aca aat cat tca tat gaa gaa gta gaa gaa
ata aca 1104Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu
Ile Thr 355 360 365
aat att cct aaa agt aca cta aaa aaa tta aaa aaa tat tat ttt taa
1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe
370 375 380
14383PRTBacillus thuringiensis 14Met Leu Asp Thr Asn Lys Val Tyr Glu Ile
Ser Asn His Ala Asn Gly 1 5 10
15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser
Leu 20 25 30 Met
Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35
40 45 Leu Phe Pro Ile Asp Asp
Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55
60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp
Lys Ile Asn Val Ser 65 70 75
80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn
85 90 95 Gly Ser
Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100
105 110 Gly Thr Gly Gln Ala Leu Gly
Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120
125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val
Gln Thr Ile Gln 130 135 140
Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145
150 155 160 Tyr Ser Pro
Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175 Gly Trp Thr Leu Val Pro Cys Ile
Met Val Asn Asp Pro Asn Ile Asp 180 185
190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu
Lys Lys Tyr 195 200 205
Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210
215 220 Glu Lys Lys Ser
Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230
235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe
Gln Ile Asn Ile Asp Ser Gly 245 250
255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu
Ile Lys 260 265 270
Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys
275 280 285 Ile Met Glu Lys
Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300 Gln Ser Met Asn Ser Ile Gly Phe
Leu Thr Ile Thr Ser Leu Glu Leu 305 310
315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln
Ile Gln Thr Ser 325 330
335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala
340 345 350 Leu Leu Leu
Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355
360 365 Asn Ile Pro Lys Ser Thr Leu Lys
Lys Leu Lys Lys Tyr Tyr Phe 370 375
380 151152DNAArtificial SequenceSynthetic DNA sequence
encoding Bacillus thuringiensis Cry35Ab1 toxin using codons
optimized for maize and Table 1 sequences are
maintainedCDS(1)..(1152) 15atg ctc gat act aat aaa gtg tat gaa ata agc
aac cat gcc aac ggg 48Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser
Asn His Ala Asn Gly1 5 10
15 cta tat gcc gca act tat ttg agt ctg gac gac agc ggt gtg agc tta
96Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu
20 25 30 atg aat aaa aac
gac gac gac att gac gac tac aac ctc aag tgg ttt 144Met Asn Lys Asn
Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35
40 45 tta ttt cct att gac gac gat caa tat
att att aca agc tac gca gca 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr
Ile Ile Thr Ser Tyr Ala Ala 50 55 60
aat aat tgc aaa gtc tgg aac gtt aat aat gat aaa ata aat
gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn
Val Ser65 70 75 80 acc
tac agc tcc acc aac tca ata caa aag tgg caa ata aaa gct aat 288Thr
Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn
85 90 95 gga tcg tcg tat gta ata
cag agt gac aat ggg aag gtc ttg aca gcg 336Gly Ser Ser Tyr Val Ile
Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100
105 110 ggc act ggt caa gct ctt gga ctc ata agg
ctc act gac gag tcc tca 384Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg
Leu Thr Asp Glu Ser Ser 115 120
125 aat aat ccc aat caa cag tgg aac ttg act tcc gtg cag acg
atc caa 432Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr
Ile Gln 130 135 140 ctt
cca cag aaa cct atc atc gat aca aaa tta aaa gat tac ccc aag 480Leu
Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys145
150 155 160tac tcg cca acc ggc aac
atc gat aat gga acg tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn
Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175 ggc tgg acc ctc gta ccc tgt att atg gtg aac
gac ccg aat atc gat 576Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn
Asp Pro Asn Ile Asp 180 185
190 aaa aat act caa att aaa acc acg ccg tat tat ata ttg aaa aaa
tac 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys
Tyr 195 200 205 caa tac
tgg cag cgc gcg gtt ggc tca aac gtc gct ctg cgg cca cat 672Gln Tyr
Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210
215 220 gaa aag aag tcc tac act tac
gaa tgg ggc aca gag atc gat cag aaa 720Glu Lys Lys Ser Tyr Thr Tyr
Glu Trp Gly Thr Glu Ile Asp Gln Lys225 230
235 240acg acc att ata aat aca tta gga ttc caa atc aat
atc gac agc gga 768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn
Ile Asp Ser Gly 245 250
255 atg aaa ttt gac atc ccg gaa gtg ggg ggc ggg acc gat gaa ata aaa
816Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys
260 265 270 acg cag ctc aac
gaa gaa tta aaa ata gag tac agt cat gaa act aaa 864Thr Gln Leu Asn
Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275
280 285 ata atg gaa aaa tat caa gag caa tct
gaa atc gat aac ccg acc gac 912Ile Met Glu Lys Tyr Gln Glu Gln Ser
Glu Ile Asp Asn Pro Thr Asp 290 295
300 caa tca atg aac tct atc ggt ttc ctt act att acc tcc
ctg gag tta 960Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser
Leu Glu Leu305 310 315
320tat aga tat aac ggc tct gag atc cgt ata atg cag att caa acc tca
1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser
325 330 335 gac aat gac act
tat aac gtc acc tct tac ccg aat cat caa caa gct 1056Asp Asn Asp Thr
Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340
345 350 tta ttg ctt ctt aca aac cac agt tat
gaa gag gtg gaa gaa ata acg 1104Leu Leu Leu Leu Thr Asn His Ser Tyr
Glu Glu Val Glu Glu Ile Thr 355 360
365 aac att cct aaa tcc aca cta aag aaa tta aaa aaa tat tat
ttt tga 1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr
Phe 370 375 380
16383PRTArtificial SequenceSynthetic Construct 16Met Leu Asp Thr Asn Lys
Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5
10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp
Ser Gly Val Ser Leu 20 25
30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp
Phe 35 40 45 Leu
Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50
55 60 Asn Asn Cys Lys Val Trp
Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70
75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp
Gln Ile Lys Ala Asn 85 90
95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110 Gly Thr
Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115
120 125 Asn Asn Pro Asn Gln Gln Trp
Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135
140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys
Asp Tyr Pro Lys 145 150 155
160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met
165 170 175 Gly Trp Thr
Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180
185 190 Lys Asn Thr Gln Ile Lys Thr Thr
Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200
205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu
Arg Pro His 210 215 220
Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225
230 235 240 Thr Thr Ile Ile
Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245
250 255 Met Lys Phe Asp Ile Pro Glu Val Gly
Gly Gly Thr Asp Glu Ile Lys 260 265
270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu
Thr Lys 275 280 285
Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300 Gln Ser Met Asn Ser
Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310
315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile
Met Gln Ile Gln Thr Ser 325 330
335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln
Ala 340 345 350 Leu
Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355
360 365 Asn Ile Pro Lys Ser Thr
Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375
380 171152DNAArtificial SequenceSynthetic DNA sequence
in accordance with the invention encoding Bacillus thuringiensis
Cry35Ab1 toxin using codons optimized for maize and with sequences
identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(1152) 17atg ctc gat act aat aaa gtg tat gaa ata tcg
aac cat gcc aac ggg 48Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser
Asn His Ala Asn Gly1 5 10
15 cta tat gcc gca act tac ctg agt ctg gac gat agt ggt gtg agc tta
96Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu
20 25 30 atg aat aaa aac
gac gac gac att gac gac tac aac ctc aag tgg ttc 144Met Asn Lys Asn
Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35
40 45 ctg ttt cct att gac gac gat cag tat
atc att aca agc tac gca gcg 192Leu Phe Pro Ile Asp Asp Asp Gln Tyr
Ile Ile Thr Ser Tyr Ala Ala 50 55 60
aat aat tgc aaa gtc tgg aac gtc aat aat gat aaa atc aat
gtt tcg 240Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn
Val Ser65 70 75 80 acc
tac agc tcc acc aac tca ata caa aag tgg caa atc aaa gct aat 288Thr
Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn
85 90 95 ggc agc tcg tac gta ata
cag agt gac aat ggg aag gtc ttg aca gcg 336Gly Ser Ser Tyr Val Ile
Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100
105 110 ggc act ggt caa gct ctt gga ctc ata agg
ctc act gac gag tcc tcg 384Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg
Leu Thr Asp Glu Ser Ser 115 120
125 aat aat ccc aat caa cag tgg aac ttg act tcc gtg cag acg
atc caa 432Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr
Ile Gln 130 135 140 ctt
cca cag aaa cct atc atc gat aca aaa tta aaa gat tac ccc aag 480Leu
Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys145
150 155 160tac tcg cca acc ggc aac
atc gat aat gga acg tct cct caa tta atg 528Tyr Ser Pro Thr Gly Asn
Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165
170 175 ggc tgg acc ctc gta ccc tgt ata atg gtg aac
gac ccg aat atc gat 576Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn
Asp Pro Asn Ile Asp 180 185
190 aaa aat act caa att aaa acc acg ccg tac tac ata ctc aaa aaa
tac 624Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys
Tyr 195 200 205 caa tac
tgg cag cgc gcg gtt ggc tca aac gtc gct ctg cgg cca cat 672Gln Tyr
Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210
215 220 gaa aag aag tcc tac act tac
gaa tgg ggc aca gag atc gat cag aaa 720Glu Lys Lys Ser Tyr Thr Tyr
Glu Trp Gly Thr Glu Ile Asp Gln Lys225 230
235 240acg acc att ata aat aca tta gga ttc caa atc aat
atc gac agc gga 768Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn
Ile Asp Ser Gly 245 250
255 atg aaa ttt gac atc ccg gaa gtg ggg ggc ggg acc gat gaa ata aaa
816Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys
260 265 270 acg cag ctc aac
gaa gaa tta aaa ata gag tac agt cat gaa act aaa 864Thr Gln Leu Asn
Glu Glu Leu Lys Ile Glu Tyr Ser His Glu Thr Lys 275
280 285 ata atg gaa aaa tat caa gag caa tct
gaa atc gat aac ccg acc gac 912Ile Met Glu Lys Tyr Gln Glu Gln Ser
Glu Ile Asp Asn Pro Thr Asp 290 295
300 caa tca atg aac tct atc ggt ttc ctt act att acc tcc
ctg gag ttg 960Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser
Leu Glu Leu305 310 315
320tac aga tat aac ggc tct gag atc cgt ata atg cag att caa acc tca
1008Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser
325 330 335 gac aat gac act
tat aac gtc acc tct tac ccg aat cat cag caa gcc 1056Asp Asn Asp Thr
Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340
345 350 ctg ctg ctt ctt aca aac cac agt tat
gaa gag gtg gaa gag ata acg 1104Leu Leu Leu Leu Thr Asn His Ser Tyr
Glu Glu Val Glu Glu Ile Thr 355 360
365 aac att cct aaa tcc aca cta aag aaa tta aaa aaa tat tat
ttc tga 1152Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr
Phe 370 375 380
18383PRTArtificial SequenceSynthetic Construct 18Met Leu Asp Thr Asn Lys
Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5
10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp
Ser Gly Val Ser Leu 20 25
30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp
Phe 35 40 45 Leu
Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50
55 60 Asn Asn Cys Lys Val Trp
Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70
75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp
Gln Ile Lys Ala Asn 85 90
95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala
100 105 110 Gly Thr
Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115
120 125 Asn Asn Pro Asn Gln Gln Trp
Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135
140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys
Asp Tyr Pro Lys 145 150 155
160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met
165 170 175 Gly Trp Thr
Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180
185 190 Lys Asn Thr Gln Ile Lys Thr Thr
Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200
205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu
Arg Pro His 210 215 220
Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225
230 235 240 Thr Thr Ile Ile
Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245
250 255 Met Lys Phe Asp Ile Pro Glu Val Gly
Gly Gly Thr Asp Glu Ile Lys 260 265
270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser His Glu
Thr Lys 275 280 285
Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290
295 300 Gln Ser Met Asn Ser
Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310
315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile
Met Gln Ile Gln Thr Ser 325 330
335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln
Ala 340 345 350 Leu
Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355
360 365 Asn Ile Pro Lys Ser Thr
Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375
380 191830DNABacillus thuringiensisCDS(1)..(1830)Native
DNA sequence encoding Bacillus thuringiensis Cry1Ab1 core toxin
19atg gat aac aat ccg aac atc aat gaa tgc att cct tat aat tgt tta
48Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1
5 10 15 agt aac cct gaa
gta gaa gta tta ggt gga gaa aga ata gaa act ggt 96Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20
25 30 tac acc cca atc gat att tcc ttg tcg
cta acg caa ttt ctt ttg agt 144Tyr Thr Pro Ile Asp Ile Ser Leu Ser
Leu Thr Gln Phe Leu Leu Ser 35 40
45 gaa ttt gtt ccc ggt gct gga ttt gtg tta gga cta gtt gat
ata ata 192Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp
Ile Ile 50 55 60 tgg
gga att ttt ggt ccc tct caa tgg gac gca ttt ctt gta caa att 240Trp
Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65
70 75 80 gaa cag tta att aac caa
aga ata gaa gaa ttc gct agg aac caa gcc 288Glu Gln Leu Ile Asn Gln
Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85
90 95 att tct aga tta gaa gga cta agc aat ctt tat
caa att tac gca gaa 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr
Gln Ile Tyr Ala Glu 100 105
110 tct ttt aga gag tgg gaa gca gat cct act aat cca gca tta aga
gaa 384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg
Glu 115 120 125 gag atg
cgt att caa ttc aat gac atg aac agt gcc ctt aca acc gct 432Glu Met
Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130
135 140 att cct ctt ttt gca gtt caa
aat tat caa gtt cct ctt tta tca gta 480Ile Pro Leu Phe Ala Val Gln
Asn Tyr Gln Val Pro Leu Leu Ser Val145 150
155 160tat gtt caa gct gca aat tta cat tta tca gtt ttg
aga gat gtt tca 528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
Arg Asp Val Ser 165 170
175 gtg ttt gga caa agg tgg gga ttt gat gcc gcg act atc aat agt cgt
576Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190 tat aat gat tta
act agg ctt att ggc aac tat aca gat tat gct gta 624Tyr Asn Asp Leu
Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195
200 205 cgc tgg tac aat acg gga tta gaa cgt
gta tgg gga ccg gat tct aga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg
Val Trp Gly Pro Asp Ser Arg 210 215
220 gat tgg gta agg tat aat caa ttt aga aga gaa tta aca
cta act gta 720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr
Leu Thr Val225 230 235
240tta gat atc gtt gct ctg ttc ccg aat tat gat agt aga aga tat cca
768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro
245 250 255 att cga aca gtt
tcc caa tta aca aga gaa att tat aca aac cca gta 816Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260
265 270 tta gaa aat ttt gat ggt agt ttt cga
ggc tcg gct cag ggc ata gaa 864Leu Glu Asn Phe Asp Gly Ser Phe Arg
Gly Ser Ala Gln Gly Ile Glu 275 280
285 aga agt att agg agt cca cat ttg atg gat ata ctt aac agt
ata acc 912Arg Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser
Ile Thr 290 295 300 atc
tat acg gat gct cat agg ggt tat tat tat tgg tca ggg cat caa 960Ile
Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln305
310 315 320ata atg gct tct cct gta
ggg ttt tcg ggg cca gaa ttc act ttt ccg 1008Ile Met Ala Ser Pro Val
Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325
330 335 cta tat gga act atg gga aat gca gct cca caa
caa cgt att gtt gct 1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln
Gln Arg Ile Val Ala 340 345
350 caa cta ggt cag ggc gtg tat aga aca tta tcg tcc act tta tat
aga 1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr
Arg 355 360 365 aga
cct ttt aat ata ggg ata aat aat caa caa cta tct gtt ctt gac 1152Arg
Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp 370
375 380 ggg aca gaa ttt gct tat
gga acc tcc tca aat ttg cca tcc gct gta 1200Gly Thr Glu Phe Ala Tyr
Gly Thr Ser Ser Asn Leu Pro Ser Ala Val385 390
395 400tac aga aaa agc gga acg gta gat tcg ctg gat
gaa ata ccg cca cag 1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp
Glu Ile Pro Pro Gln 405 410
415 aat aac aac gtg cca cct agg caa gga ttt agt cat cga tta agc cat
1296Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430 gtt tca atg ttt
cgt tca ggc ttt agt aat agt agt gta agt ata ata 1344Val Ser Met Phe
Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile 435
440 445 aga gct cct atg ttc tct tgg ata cat
cgt agt gct gaa ttt aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His
Arg Ser Ala Glu Phe Asn Asn 450 455
460 ata att cct tca tca caa att aca caa ata cct tta aca
aaa tct act 1440Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr
Lys Ser Thr465 470 475
480aat ctt ggc tct gga act tct gtc gtt aaa gga cca gga ttt aca gga
1488Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly
485 490 495 gga gat att ctt
cga aga act tca cct ggc cag att tca acc tta aga 1536Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg 500
505 510 gta aat att act gca cca tta tca caa
aga tat cgg gta aga att cgc 1584Val Asn Ile Thr Ala Pro Leu Ser Gln
Arg Tyr Arg Val Arg Ile Arg 515 520
525 tac gct tct acc aca aat tta caa ttc cat aca tca att gac
gga aga 1632Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp
Gly Arg 530 535 540 cct
att aat cag ggg aat ttt tca gca act atg agt agt ggg agt aat 1680Pro
Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn545
550 555 560tta cag tcc gga agc ttt
agg act gta ggt ttt act act ccg ttt aac 1728Leu Gln Ser Gly Ser Phe
Arg Thr Val Gly Phe Thr Thr Pro Phe Asn 565
570 575 ttt tca aat gga tca agt gta ttt acg tta agt
gct cat gtc ttc aat 1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser
Ala His Val Phe Asn 580 585
590 tca ggc aat gaa gtt tat ata gat cga att gaa ttt gtt ccg gca
gaa 1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala
Glu 595 600 605 gta acc
1830Val Thr
61020610PRTBacillus thuringiensis 20Met Asp Asn Asn Pro Asn Ile Asn Glu
Cys Ile Pro Tyr Asn Cys Leu 1 5 10
15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu
Thr Gly 20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45 Glu Phe Val Pro
Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50
55 60 Trp Gly Ile Phe Gly Pro Ser Gln
Trp Asp Ala Phe Leu Val Gln Ile 65 70
75 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala
Arg Asn Gln Ala 85 90
95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110 Ser Phe Arg
Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115
120 125 Glu Met Arg Ile Gln Phe Asn Asp
Met Asn Ser Ala Leu Thr Thr Ala 130 135
140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu
Leu Ser Val 145 150 155
160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175 Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180
185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly
Asn Tyr Thr Asp Tyr Ala Val 195 200
205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp
Ser Arg 210 215 220
Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225
230 235 240 Leu Asp Ile Val Ala
Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245
250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu
Ile Tyr Thr Asn Pro Val 260 265
270 Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile
Glu 275 280 285 Arg
Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290
295 300 Ile Tyr Thr Asp Ala His
Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310
315 320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro
Glu Phe Thr Phe Pro 325 330
335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350 Gln Leu
Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355
360 365 Arg Pro Phe Asn Ile Gly Ile
Asn Asn Gln Gln Leu Ser Val Leu Asp 370 375
380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu
Pro Ser Ala Val 385 390 395
400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415 Asn Asn Asn
Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420
425 430 Val Ser Met Phe Arg Ser Gly Phe
Ser Asn Ser Ser Val Ser Ile Ile 435 440
445 Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu
Phe Asn Asn 450 455 460
Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465
470 475 480 Asn Leu Gly Ser
Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485
490 495 Gly Asp Ile Leu Arg Arg Thr Ser Pro
Gly Gln Ile Ser Thr Leu Arg 500 505
510 Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg
Ile Arg 515 520 525
Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530
535 540 Pro Ile Asn Gln Gly
Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn 545 550
555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly
Phe Thr Thr Pro Phe Asn 565 570
575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe
Asn 580 585 590 Ser
Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605 Val Thr 610
211830DNAArtificial SequenceSynthetic DNA sequence encoding Bacillus
thuringiensis Cry1Ab1 core toxin using codons optimized for maize
and Table 1 sequences are maintainedCDS(1)..(1830) 21atg gat aac aac ccg
aac atc aat gag tgc atc ccg tat aac tgt ctc 48Met Asp Asn Asn Pro
Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15 agt aac cct gaa gtg gag gtc tta ggt ggc
gaa cgc atc gaa act ggt 96Ser Asn Pro Glu Val Glu Val Leu Gly Gly
Glu Arg Ile Glu Thr Gly 20 25
30 tac acc cca atc gac att agc ttg tcg ttg acg cag ttc ctt ttg
tcc 144Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu
Ser 35 40 45 gag ttc
gtg ccc ggt gcg ggt ttc gtg ctg ggg cta gtt gat ata atc 192Glu Phe
Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50
55 60 tgg gga atc ttt ggt ccc tct
cag tgg gac gcc ttt ctt gtg caa att 240Trp Gly Ile Phe Gly Pro Ser
Gln Trp Asp Ala Phe Leu Val Gln Ile65 70
75 80 gag cag cta att aac caa aga ata gaa gag ttc gcg
agg aac caa gcc 288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala
Arg Asn Gln Ala 85 90 95
att tcc aga ctg gag gga cta agc aac ctt tat caa atc tac gcg gag
336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110 tct ttt agg gag
tgg gag gca gat cct acg aac ccg gca ctg cgc gaa 384Ser Phe Arg Glu
Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115
120 125 gag atg cgt att cag ttc aac gac atg
aac agt gcc ctt aca acc gct 432Glu Met Arg Ile Gln Phe Asn Asp Met
Asn Ser Ala Leu Thr Thr Ala 130 135
140 att ccc ctt ttc gca gtt caa aat tac caa gtt ccc ctt
ctc tca gtg 480Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu
Leu Ser Val145 150 155
160tac gtt caa gcc gca aat tta cac cta agc gtt ctc cgc gat gtg tca
528Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175 gtg ttt ggc cag
agg tgg gga ttt gat gcc gcc act atc aat agt cgt 576Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180
185 190 tat aat gat ctg acg agg ctt atc ggc
aac tat acc gac tat gct gtc 624Tyr Asn Asp Leu Thr Arg Leu Ile Gly
Asn Tyr Thr Asp Tyr Ala Val 195 200
205 cgc tgg tac aat acg gga tta gag cgg gtc tgg ggt ccg gat
tcc cga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp
Ser Arg 210 215 220 gac
tgg gtg cgc tac aat caa ttc cgc cgc gaa tta acc ctc act gtc 720Asp
Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val225
230 235 240ctc gac atc gtg gcg ctg
ttc ccg aac tac gac agt agg aga tac cca 768Leu Asp Ile Val Ala Leu
Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245
250 255 atc cgc aca gtt tcc caa tta acg cgg gaa att
tac acc aac cca gtc 816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile
Tyr Thr Asn Pro Val 260 265
270 ctg gag aat ttt gac ggg agc ttc cga ggc tcg gct caa ggc ata
gaa 864Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile
Glu 275 280 285 cgc agc
att agg tcg cca cac ttg atg gat atc ctt aac agc atc acc 912Arg Ser
Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290
295 300 atc tac acg gat gcc cat agg
ggt tac tac tac tgg tcg ggg cat caa 960Ile Tyr Thr Asp Ala His Arg
Gly Tyr Tyr Tyr Trp Ser Gly His Gln305 310
315 320ata atg gct tct cct gtc ggg ttt tcg ggg cca gag
ttc acc ttc ccg 1008Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu
Phe Thr Phe Pro 325 330
335 ctc tac ggc act atg gga aat gcc gcg cca caa caa cgt atc gtc gct
1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350 caa cta ggt caa
ggc gtg tac cgg aca ctg tcg tcc act ctc tat cgg 1104Gln Leu Gly Gln
Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355
360 365 cgg cct ttc aat ata ggg ata aat aat
caa cag ttg tct gtg ctg gac 1152Arg Pro Phe Asn Ile Gly Ile Asn Asn
Gln Gln Leu Ser Val Leu Asp 370 375
380 ggg aca gag ttt gct tac gga acc tca agc aac ttg cca
tcc gct gta 1200Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro
Ser Ala Val385 390 395
400tac aga aaa agc ggc acg gtg gac tcg ctg gat gaa atc ccg ccc cag
1248Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415 aat aac aac gtg
ccc cct cgg caa ggc ttc agt cat cga ctg agc cac 1296Asn Asn Asn Val
Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His 420
425 430 gtt agc atg ttc cgt tcg ggc ttc agc
aac tcc tcc gta agt ata ata 1344Val Ser Met Phe Arg Ser Gly Phe Ser
Asn Ser Ser Val Ser Ile Ile 435 440
445 aga gca cct atg ttc agc tgg ata cat cgt tcc gcc gag ttt
aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe
Asn Asn 450 455 460 ata
att ccc tcc tct caa atc aca cag atc cct ctg aca aag tct act 1440Ile
Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr465
470 475 480aat ctt ggc tct ggg act
tct gtc gtt aag ggg cct ggc ttt acg ggc 1488Asn Leu Gly Ser Gly Thr
Ser Val Val Lys Gly Pro Gly Phe Thr Gly 485
490 495 ggc gat att ctg cgg aga act tca cct ggc cag
att tcc acc ctg cgc 1536Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln
Ile Ser Thr Leu Arg 500 505
510 gtg aat atc acc gcg cca ttg tca caa cgt tac cgc gtg cgg att
cgc 1584Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile
Arg 515 520 525 tac gct
tct acc aca aac ctc cag ttc cat aca tct att gac ggc aga 1632Tyr Ala
Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530
535 540 ccc att aat caa ggg aat ttc
tcc gcc acg atg tcg tcc ggc tcc aat 1680Pro Ile Asn Gln Gly Asn Phe
Ser Ala Thr Met Ser Ser Gly Ser Asn545 550
555 560ctc cag tcc gga agt ttc cgc acc gta ggt ttt act
acc ccg ttc aac 1728Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr
Thr Pro Phe Asn 565 570
575 ttt tca aac ggc tca agt gtg ttt acg ctg tcc gct cat gtg ttc aac
1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590 tct ggc aat gag
gtt tat atc gac cgg att gag ttc gtc ccg gca gaa 1824Ser Gly Asn Glu
Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605 gtc acc
1830Val Thr 61022610PRTArtificial
SequenceSynthetic Construct 22Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile
Pro Tyr Asn Cys Leu 1 5 10
15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30 Tyr Thr
Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35
40 45 Glu Phe Val Pro Gly Ala Gly
Phe Val Leu Gly Leu Val Asp Ile Ile 50 55
60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile 65 70 75
80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95 Ile Ser Arg
Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100
105 110 Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu 115 120
125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu
Thr Thr Ala 130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145
150 155 160 Tyr Val Gln Ala
Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165
170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp
Ala Ala Thr Ile Asn Ser Arg 180 185
190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210
215 220 Asp Trp Val Arg Tyr
Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230
235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr
Asp Ser Arg Arg Tyr Pro 245 250
255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro
Val 260 265 270 Leu
Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275
280 285 Arg Ser Ile Arg Ser Pro
His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295
300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr
Trp Ser Gly His Gln 305 310 315
320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335 Leu Tyr
Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340
345 350 Gln Leu Gly Gln Gly Val Tyr
Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360
365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp 370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385
390 395 400 Tyr Arg Lys
Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405
410 415 Asn Asn Asn Val Pro Pro Arg Gln
Gly Phe Ser His Arg Leu Ser His 420 425
430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val
Ser Ile Ile 435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450
455 460 Ile Ile Pro Ser
Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470
475 480 Asn Leu Gly Ser Gly Thr Ser Val Val
Lys Gly Pro Gly Phe Thr Gly 485 490
495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr
Leu Arg 500 505 510
Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg
515 520 525 Tyr Ala Ser Thr
Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530
535 540 Pro Ile Asn Gln Gly Asn Phe Ser
Ala Thr Met Ser Ser Gly Ser Asn 545 550
555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr
Thr Pro Phe Asn 565 570
575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590 Ser Gly Asn
Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605 Val Thr 610
231830DNAArtificial SequenceSynthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry1Ab1 core toxin using
codons optimized for maize and with sequences identified in Table
2 removed and Table 1 sequences are maintainedCDS(1)..(1830) 23atg gat
aac aac ccg aac atc aat gag tgc atc ccg tat aac tgt ctc 48Met Asp
Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1
5 10 15 agt aac cct gaa
gtg gag gtc tta ggt ggc gaa cgc atc gaa act ggt 96Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20
25 30 tac acc cca atc gac att
agc ttg tcg ttg acg cag ttc ctc ttg tcc 144Tyr Thr Pro Ile Asp Ile
Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35
40 45 gag ttc gtg ccc ggt gcg ggt ttc
gtg ctg ggg cta gtt gat ata atc 192Glu Phe Val Pro Gly Ala Gly Phe
Val Leu Gly Leu Val Asp Ile Ile 50 55
60 tgg gga atc ttt ggt ccc tct cag tgg gac
gcc ttt ctt gtg caa att 240Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp
Ala Phe Leu Val Gln Ile 65 70 75
80 gag cag cta att aac caa aga ata gaa gag ttc gcg
agg aac caa gcc 288Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala
Arg Asn Gln Ala 85 90
95 att tcc aga ctg gag gga cta agc aac ctt tat caa atc tac
gcg gag 336Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr
Ala Glu 100 105 110
tct ttt agg gag tgg gag gca gat cct acg aac ccg gca ctg cgc gaa
384Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125 gag
atg cgt att cag ttc aac gac atg aac agt gcc ctt aca acc gct 432Glu
Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130
135 140 att ccc ctt
ttc gca gtt caa aat tac caa gtt ccc ctt ctc tca gtg 480Ile Pro Leu
Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145
150 155 160 tac gtt caa gcc gca
aat tta cac cta agc gtt ctc cgc gat gtg tca 528Tyr Val Gln Ala Ala
Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165
170 175 gtg ttc ggc cag agg tgg gga
ttt gat gcc gcc act atc aat agt cgt 576Val Phe Gly Gln Arg Trp Gly
Phe Asp Ala Ala Thr Ile Asn Ser Arg 180
185 190 tat aat gat ctg acg agg ctt atc ggc
aac tat acc gac tat gct gtc 624Tyr Asn Asp Leu Thr Arg Leu Ile Gly
Asn Tyr Thr Asp Tyr Ala Val 195 200
205 cgc tgg tac aat acg gga tta gag cgg gtc tgg
ggt ccg gat tcc cga 672Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp
Gly Pro Asp Ser Arg 210 215 220
gac tgg gtg cgc tac aat caa ttc cgc cgc gaa tta acc
ctc act gtc 720Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr
Leu Thr Val 225 230 235
240 ctc gac atc gtg gcg ctg ttc ccg aac tac gac agt agg aga tac
cca 768Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr
Pro 245 250 255
atc cgc aca gtt tcc caa tta acg cgg gaa att tac acc aac cca gtc
816Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270 ctg
gag aat ttt gac ggg agc ttc cga ggc tcg gct caa ggc ata gaa 864Leu
Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285 cgc agc att
agg tcg cca cac ttg atg gat atc ctt aac agc atc acc 912Arg Ser Ile
Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290
295 300 atc tac acg gat gcc
cat agg ggt tac tac tac tgg tcg ggg cat caa 960Ile Tyr Thr Asp Ala
His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310
315 320 ata atg gct tct cct gtc ggg
ttt tcg ggg cca gag ttc acc ttc ccg 1008Ile Met Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe Thr Phe Pro 325
330 335 ctc tac ggc act atg gga aat gcc gcg cca
caa caa cgt atc gtc gct 1056Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro
Gln Gln Arg Ile Val Ala 340 345
350 caa cta ggt caa ggc gtg tac cgg aca ctg tcg tcc act
ctc tat cgg 1104Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr
Leu Tyr Arg 355 360 365
cgg cct ttc aat ata ggg atc aat aat caa cag ttg tct gtg ctg gac
1152Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380 ggg
aca gag ttt gct tac gga acc tca agc aac ttg cca tcc gct gta 1200Gly
Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385
390 395 400 tac aga aaa agc
ggc acg gtg gac tcg ctg gat gaa atc ccg ccc cag 1248Tyr Arg Lys Ser
Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405
410 415 aat aac aac gtg ccc cct cgg
caa ggc ttc agt cat cga ctg agc cac 1296Asn Asn Asn Val Pro Pro Arg
Gln Gly Phe Ser His Arg Leu Ser His 420
425 430 gtt agc atg ttc cgt tcg ggc ttc agc
aac tcc tcc gta agt atc ata 1344Val Ser Met Phe Arg Ser Gly Phe Ser
Asn Ser Ser Val Ser Ile Ile 435 440
445 aga gca cct atg ttc agc tgg ata cat cgt tcc gcc
gag ttc aat aat 1392Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
Glu Phe Asn Asn 450 455 460
ata att ccc tcc tct caa atc aca cag atc cct ctg aca aag tct
act 1440Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser
Thr 465 470 475 480
aat ctt ggc tct ggg act tct gtc gtt aag ggg cct ggc ttt acg ggc
1488Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly
485 490 495 ggc gat
att ctg cgg aga act tca cct ggc cag att tcc acc ctg cgc 1536Gly Asp
Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg
500 505 510 gtg aat atc acc
gcg cca ttg tca caa cgt tac cgc gtg cgg att cgc 1584Val Asn Ile Thr
Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg 515
520 525 tac gct tct acc aca aac ctc
cag ttc cat aca tct att gac ggc aga 1632Tyr Ala Ser Thr Thr Asn Leu
Gln Phe His Thr Ser Ile Asp Gly Arg 530 535
540 ccc att aat caa ggg aat ttc tcc gcc acg
atg tcg tcc ggc tcc aat 1680Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr
Met Ser Ser Gly Ser Asn 545 550 555
560 ctc cag tcc gga agt ttc cgc acc gta ggt ttt act acc
ccg ttc aac 1728Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr
Pro Phe Asn 565 570
575 ttt tca aac ggc tca agt gtg ttt acg ctg tcc gct cat gtg ttc
aac 1776Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe
Asn 580 585 590
tct ggc aat gag gtt tac atc gac cgg att gag ttc gtc ccg gca gaa
1824Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu
595 600 605 gtc acc
1830Val Thr
610
24610PRTArtificial
SequenceSynthetic Construct 24Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile
Pro Tyr Asn Cys Leu 1 5 10
15 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30 Tyr Thr
Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35
40 45 Glu Phe Val Pro Gly Ala Gly
Phe Val Leu Gly Leu Val Asp Ile Ile 50 55
60 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile 65 70 75
80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95 Ile Ser Arg
Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100
105 110 Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu 115 120
125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu
Thr Thr Ala 130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145
150 155 160 Tyr Val Gln Ala
Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165
170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp
Ala Ala Thr Ile Asn Ser Arg 180 185
190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210
215 220 Asp Trp Val Arg Tyr
Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val 225 230
235 240 Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr
Asp Ser Arg Arg Tyr Pro 245 250
255 Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro
Val 260 265 270 Leu
Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275
280 285 Arg Ser Ile Arg Ser Pro
His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295
300 Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr
Trp Ser Gly His Gln 305 310 315
320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335 Leu Tyr
Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala 340
345 350 Gln Leu Gly Gln Gly Val Tyr
Arg Thr Leu Ser Ser Thr Leu Tyr Arg 355 360
365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp 370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val 385
390 395 400 Tyr Arg Lys
Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln 405
410 415 Asn Asn Asn Val Pro Pro Arg Gln
Gly Phe Ser His Arg Leu Ser His 420 425
430 Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val
Ser Ile Ile 435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn 450
455 460 Ile Ile Pro Ser
Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr 465 470
475 480 Asn Leu Gly Ser Gly Thr Ser Val Val
Lys Gly Pro Gly Phe Thr Gly 485 490
495 Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr
Leu Arg 500 505 510
Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg
515 520 525 Tyr Ala Ser Thr
Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg 530
535 540 Pro Ile Asn Gln Gly Asn Phe Ser
Ala Thr Met Ser Ser Gly Ser Asn 545 550
555 560 Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr
Thr Pro Phe Asn 565 570
575 Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn
580 585 590 Ser Gly Asn
Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu 595
600 605 Val Thr 610
251866DNABacillus thuringiensisCDS(1)..(1866)Native DNA sequence encoding
Bacillus thuringiensis Cry1Ca core toxin 25atg gat aac aat ccg aac
atc aat gaa tgc atc ccg tac aac tgc ctg 48Met Asp Asn Asn Pro Asn
Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu1 5
10 15 agc aac ccg gaa gaa gtg ctg ttg gat gga gaa
cgg ata tca act ggt 96Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu
Arg Ile Ser Thr Gly 20 25 30
aat tca tca att gat att tct ctg tca ctt gtt cag ttt ctg gta tct
144Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser
35 40 45 aac
ttc gtc cca ggc gga gga ttc ctg gtt gga tta ata gat ttt gta 192Asn
Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50
55 60 tgg gga ata
gtt ggc cct tct caa tgg gat gca ttt cta gta caa att 240Trp Gly Ile
Val Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65
70 75 80 gaa caa tta att aat
gaa aga ata gct gaa ttt gct agg aat gct gct 288Glu Gln Leu Ile Asn
Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala 85
90 95 att gct aat tta gaa gga tta
gga aac aat ttc aat ata tat gtg gaa 336Ile Ala Asn Leu Glu Gly Leu
Gly Asn Asn Phe Asn Ile Tyr Val Glu 100
105 110 gca ttt aaa gaa tgg gaa gaa gat cct
aag aat cca gca acc agg acc 384Ala Phe Lys Glu Trp Glu Glu Asp Pro
Lys Asn Pro Ala Thr Arg Thr 115 120
125 aga gta att gat cgc ttt cgt ata ctt gat ggg
cta ctt gaa agg gac 432Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly
Leu Leu Glu Arg Asp 130 135 140
att cct tcg ttt cga att tct gga ttt gaa gta ccc ctt
tta tcc gtt 480Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu
Leu Ser Val 145 150 155
160 tat gct caa gcg gcc aat ctg cat cta gct ata tta aga gat tct
gta 528Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser
Val 165 170 175
att ttt gga gaa aga tgg gga ttg aca acg ata aat gtc aat gaa aac
576Ile Phe Gly Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn
180 185 190 tat
aat aga cta att agg cat att gat gaa tat gct gat cac tgt gca 624Tyr
Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His Cys Ala
195 200 205 aat acg tat
aat cgg gga tta aat aat tta ccg aaa tct acg tat caa 672Asn Thr Tyr
Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220 gat tgg ata aca tat
aat cga ttg cgg aga gac tta aca ttg act gta 720Asp Trp Ile Thr Tyr
Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230
235 240 tta gat atc gcc gct ttc ttt
cca aac tat gac aat agg aga tat cca 768Leu Asp Ile Ala Ala Phe Phe
Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245
250 255 att cag cca gtt ggt caa cta aca agg
gaa gtt tat acg gac cca tta 816Ile Gln Pro Val Gly Gln Leu Thr Arg
Glu Val Tyr Thr Asp Pro Leu 260 265
270 att aat ttt aat cca cag tta cag tct gta gct
caa tta cct act ttt 864Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala
Gln Leu Pro Thr Phe 275 280
285 aac gtt atg gag aac agc gca att aga aat cct cat tta
ttt gat ata 912Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu
Phe Asp Ile 290 295 300
ttg aat aat ctt aca atc ttt acg gat tgg ttt agt gtt gga cgc
aat 960Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe Ser Val Gly Arg
Asn 305 310 315 320
ttt tat tgg gga gga cat cga gta ata tct agc ctt ata gga ggt ggt
1008Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335 aac ata
aca tct cct ata tat gga aga gag gcg aac cag gag cct cca 1056Asn Ile
Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro
340 345 350 aga tcc ttt act
ttt aat gga ccg gta ttt agg act tta tca aat cct 1104Arg Ser Phe Thr
Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355
360 365 act tta cga tta tta cag caa
cct tgg cca gcg cca cca ttt aat tta 1152Thr Leu Arg Leu Leu Gln Gln
Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375
380 cgt ggt gtt gaa gga gta gaa ttt tct aca
cct aca aat agc ttt acg 1200Arg Gly Val Glu Gly Val Glu Phe Ser Thr
Pro Thr Asn Ser Phe Thr 385 390 395
400 tat cga gga aga ggt acg gtt gat tct tta act gaa ttg
ccg cct gag 1248Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu
Pro Pro Glu 405 410
415 gat aat agt gtg cca cct cgc gaa gga tat agt cat cgt tta tgt
cat 1296Asp Asn Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys
His 420 425 430
gca act ttt gtt caa aga tct gga aca cct ttt tta aca act ggt gta
1344Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr Thr Gly Val
435 440 445 gta ttt
tct tgg acg cat cgt agt gca act ctt aca aat aca att gat 1392Val Phe
Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450
455 460 cca gag aga att aat
caa ata cct tta gtg aaa gga ttt aga gtt tgg 1440Pro Glu Arg Ile Asn
Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470
475 480 ggg ggc acc tct gtc att aca gga
cca gga ttt aca gga ggg gat atc 1488Gly Gly Thr Ser Val Ile Thr Gly
Pro Gly Phe Thr Gly Gly Asp Ile 485
490 495 ctt cga aga aat acc ttt ggt gat ttt gta
tct cta caa gtc aat att 1536Leu Arg Arg Asn Thr Phe Gly Asp Phe Val
Ser Leu Gln Val Asn Ile 500 505
510 aat tca cca att acc caa aga tac cgt tta aga ttt cgt
tac gct tcc 1584Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
Tyr Ala Ser 515 520 525
agt agg gat gca cga gtt ata gta tta aca gga gcg gca tcc aca gga
1632Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly
530 535 540 gtg
gga ggc caa gtt agt gta aat atg cct ctt cag aaa act atg gaa 1680Val
Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545
550 555 560 ata ggg gag aac
tta aca tct aga aca ttt aga tat acc gat ttt agt 1728Ile Gly Glu Asn
Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 565
570 575 aat cct ttt tca ttt aga gct
aat cca gat ata att ggg ata agt gaa 1776Asn Pro Phe Ser Phe Arg Ala
Asn Pro Asp Ile Ile Gly Ile Ser Glu 580
585 590 caa cct cta ttt ggt gca ggt tct att
agt agc ggt gaa ctt tat ata 1824Gln Pro Leu Phe Gly Ala Gly Ser Ile
Ser Ser Gly Glu Leu Tyr Ile 595 600
605 gat aaa att gaa att att cta gca gat gca aca ttt
gaa taa 1866Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe
Glu 610 615 620
26621PRTBacillus thuringiensis 26Met Asp Asn Asn Pro Asn
Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5
10 15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu
Arg Ile Ser Thr Gly 20 25
30 Asn Ser Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val
Ser 35 40 45 Asn
Phe Val Pro Gly Gly Gly Phe Leu Val Gly Leu Ile Asp Phe Val 50
55 60 Trp Gly Ile Val Gly Pro
Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 70
75 80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe
Ala Arg Asn Ala Ala 85 90
95 Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu
100 105 110 Ala Phe
Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr 115
120 125 Arg Val Ile Asp Arg Phe Arg
Ile Leu Asp Gly Leu Leu Glu Arg Asp 130 135
140 Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro
Leu Leu Ser Val 145 150 155
160 Tyr Ala Gln Ala Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val
165 170 175 Ile Phe Gly
Glu Arg Trp Gly Leu Thr Thr Ile Asn Val Asn Glu Asn 180
185 190 Tyr Asn Arg Leu Ile Arg His Ile
Asp Glu Tyr Ala Asp His Cys Ala 195 200
205 Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser
Thr Tyr Gln 210 215 220
Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225
230 235 240 Leu Asp Ile Ala
Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr Pro 245
250 255 Ile Gln Pro Val Gly Gln Leu Thr Arg
Glu Val Tyr Thr Asp Pro Leu 260 265
270 Ile Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro
Thr Phe 275 280 285
Asn Val Met Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300 Leu Asn Asn Leu Thr
Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310
315 320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser
Ser Leu Ile Gly Gly Gly 325 330
335 Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro
Pro 340 345 350 Arg
Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu Ser Asn Pro 355
360 365 Thr Leu Arg Leu Leu Gln
Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu 370 375
380 Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro
Thr Asn Ser Phe Thr 385 390 395
400 Tyr Arg Gly Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu
405 410 415 Asp Asn
Ser Val Pro Pro Arg Glu Gly Tyr Ser His Arg Leu Cys His 420
425 430 Ala Thr Phe Val Gln Arg Ser
Gly Thr Pro Phe Leu Thr Thr Gly Val 435 440
445 Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr
Asn Thr Ile Asp 450 455 460
Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465
470 475 480 Gly Gly Thr
Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 485
490 495 Leu Arg Arg Asn Thr Phe Gly Asp
Phe Val Ser Leu Gln Val Asn Ile 500 505
510 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
Tyr Ala Ser 515 520 525
Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530
535 540 Val Gly Gly Gln
Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 545 550
555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr
Phe Arg Tyr Thr Asp Phe Ser 565 570
575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile
Ser Glu 580 585 590
Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile
595 600 605 Asp Lys Ile Glu
Ile Ile Leu Ala Asp Ala Thr Phe Glu 610 615
620 271866DNAArtificial SequenceSynthetic DNA sequence encoding
Bacillus thuringiensis Cry1Ca core toxin using codons optimized for
maize and Table 1 sequences are maintainedCDS(1)..(1866) 27atg gat
aac aat ccg aac atc aat gag tgc atc ccg tac aac tgc ctg 48Met Asp
Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1
5 10 15 agc aac ccg gaa
gaa gtg ctg ttg gat gga gaa cgg ata tca act ggc 96Ser Asn Pro Glu
Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20
25 30 aat tca tcc att gat att
tct ctg tca ctt gtt cag ttt ctg gtg tct 144Asn Ser Ser Ile Asp Ile
Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35
40 45 aac ttc gtc ccc ggc gga gga ttc
ctg gtt gga tta ata gat ttt gta 192Asn Phe Val Pro Gly Gly Gly Phe
Leu Val Gly Leu Ile Asp Phe Val 50 55
60 tgg gga ata gtt ggc cct tct caa tgg gac
gca ttt cta gta caa att 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp
Ala Phe Leu Val Gln Ile 65 70 75
80 gaa caa tta att aat gaa aga ata gct gaa ttt gct
agg aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala
Arg Asn Ala Ala 85 90
95 att gct aat tta gaa gga tta gga aac aat ttc aat ata tat
gtg gaa 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr
Val Glu 100 105 110
gca ttt aag gaa tgg gaa gaa gat cct aag aat cca gca acg agg acc
384Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr
115 120 125 aga
gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac 432Arg
Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130
135 140 att cct tcg
ttt cga att tct gga ttt gaa gta ccc ctt tta tcc gtt 480Ile Pro Ser
Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145
150 155 160 tat gct caa gcg gcc
aat ctg cat cta gct ata tta aga gat tct gta 528Tyr Ala Gln Ala Ala
Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165
170 175 att ttt gga gaa aga tgg gga
ttg aca acg ata aat gtc aat gaa aac 576Ile Phe Gly Glu Arg Trp Gly
Leu Thr Thr Ile Asn Val Asn Glu Asn 180
185 190 tat aat aga cta att agg cat att gat
gaa tat gct gat cac tgt gca 624Tyr Asn Arg Leu Ile Arg His Ile Asp
Glu Tyr Ala Asp His Cys Ala 195 200
205 aat acg tat aat cgg gga tta aat aat tta ccg
aaa tct acg tat caa 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro
Lys Ser Thr Tyr Gln 210 215 220
gat tgg ata aca tat aat cga ttg cgg aga gac tta aca
ttg act gta 720Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr
Leu Thr Val 225 230 235
240 tta gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat
cca 768Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr
Pro 245 250 255
att cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta
816Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu
260 265 270 att
aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt 864Ile
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe
275 280 285 aac gtt atg
gag aac agc gca att aga aat cct cat tta ttt gat ata 912Asn Val Met
Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300 ttg aat aat ctt aca
atc ttt acg gat tgg ttt agt gtt gga cgc aat 960Leu Asn Asn Leu Thr
Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310
315 320 ttt tat tgg gga gga cat cga
gta ata tct agc ctt ata gga ggt ggg 1008Phe Tyr Trp Gly Gly His Arg
Val Ile Ser Ser Leu Ile Gly Gly Gly 325
330 335 aac atc aca tcg cct ata tat gga aga gag
gcg aac caa gag cct cca 1056Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu
Ala Asn Gln Glu Pro Pro 340 345
350 aga tcc ttt act ttt aat gga ccc gtg ttt agg act tta
tca aat cct 1104Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu
Ser Asn Pro 355 360 365
act tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta
1152Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu
370 375 380 cgt
ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg 1200Arg
Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385
390 395 400 tat cga gga aga
ggg acg gtt gat tct tta act gaa ttg ccg cct gag 1248Tyr Arg Gly Arg
Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405
410 415 gat aat agt gtg cca cct cgc
gaa gga tat agt cat cgt tta tgt cat 1296Asp Asn Ser Val Pro Pro Arg
Glu Gly Tyr Ser His Arg Leu Cys His 420
425 430 gca act ttt gtt caa aga tcg gga aca
cct ttt tta aca act ggt gta 1344Ala Thr Phe Val Gln Arg Ser Gly Thr
Pro Phe Leu Thr Thr Gly Val 435 440
445 gta ttt tct tgg acg cat cgt agt gca act ctt aca
aat aca atc gac 1392Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr
Asn Thr Ile Asp 450 455 460
cca gag aga att aat caa ata cct tta gtg aag gga ttt aga gtt
tgg 1440Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val
Trp 465 470 475 480
ggg ggc acc tct gtc att acc gga ccc gga ttt acc gga ggg gat atc
1488Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
485 490 495 ctt cga
aga aat acc ttt ggt gat ttt gta tct cta caa gtc aac att 1536Leu Arg
Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile
500 505 510 aat tca cca att
acc caa aga tac cgt tta aga ttt cgt tac gct tcc 1584Asn Ser Pro Ile
Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515
520 525 agt agg gat gca cga gtt ata
gta tta acg gga gcg gca tcc acc gga 1632Ser Arg Asp Ala Arg Val Ile
Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535
540 gtg gga ggc caa gtt agt gta aat atg cct
ctt cag aaa act atg gaa 1680Val Gly Gly Gln Val Ser Val Asn Met Pro
Leu Gln Lys Thr Met Glu 545 550 555
560 ata ggg gag aac tta aca tcc aga aca ttt aga tat acc
gat ttt agt 1728Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr
Asp Phe Ser 565 570
575 aat cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt
gaa 1776Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser
Glu 580 585 590
caa cct cta ttt ggg gcg ggt tct att agt agc ggt gaa ctt tat ata
1824Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile
595 600 605 gat aaa
att gaa att att cta gca gat gca aca ttt gaa tga 1866Asp Lys
Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610
615 620 28621PRTArtificial
SequenceSynthetic Construct 28Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile
Pro Tyr Asn Cys Leu 1 5 10
15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
20 25 30 Asn Ser
Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35
40 45 Asn Phe Val Pro Gly Gly Gly
Phe Leu Val Gly Leu Ile Asp Phe Val 50 55
60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile 65 70 75
80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95 Ile Ala Asn
Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100
105 110 Ala Phe Lys Glu Trp Glu Glu Asp
Pro Lys Asn Pro Ala Thr Arg Thr 115 120
125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu
Glu Arg Asp 130 135 140
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145
150 155 160 Tyr Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165
170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr
Thr Ile Asn Val Asn Glu Asn 180 185
190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His
Cys Ala 195 200 205
Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220 Asp Trp Ile Thr Tyr
Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230
235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr
Asp Asn Arg Arg Tyr Pro 245 250
255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro
Leu 260 265 270 Ile
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275
280 285 Asn Val Met Glu Asn Ser
Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295
300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe
Ser Val Gly Arg Asn 305 310 315
320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335 Asn Ile
Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340
345 350 Arg Ser Phe Thr Phe Asn Gly
Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360
365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro
Pro Phe Asn Leu 370 375 380
Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385
390 395 400 Tyr Arg Gly
Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405
410 415 Asp Asn Ser Val Pro Pro Arg Glu
Gly Tyr Ser His Arg Leu Cys His 420 425
430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr
Thr Gly Val 435 440 445
Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450
455 460 Pro Glu Arg Ile
Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470
475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro
Gly Phe Thr Gly Gly Asp Ile 485 490
495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val
Asn Ile 500 505 510
Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser
515 520 525 Ser Arg Asp Ala
Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530
535 540 Val Gly Gly Gln Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu 545 550
555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr
Thr Asp Phe Ser 565 570
575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
580 585 590 Gln Pro Leu
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595
600 605 Asp Lys Ile Glu Ile Ile Leu Ala
Asp Ala Thr Phe Glu 610 615 620
291866DNAArtificial SequenceSynthetic DNA sequence in accordance with the
invention encoding Bacillus thuringiensis Cry1Ca core toxin using
codons optimized for maize and with sequences identified in Table
2 removed and Table 1 sequences are maintainedCDS(1)..(1866) 29atg gat
aac aat ccg aac atc aat gag tgc atc ccg tac aac tgc ctg 48Met Asp
Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1
5 10 15 agc aac ccg gaa
gaa gtg ctg ttg gat gga gaa cgg ata tca act ggc 96Ser Asn Pro Glu
Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly 20
25 30 aat tca tcc att gac att
tct ctg tca ctt gtt cag ttt ctg gtg tct 144Asn Ser Ser Ile Asp Ile
Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35
40 45 aac ttc gtc ccc ggc gga gga ttc
ctg gtt gga tta ata gat ttc gta 192Asn Phe Val Pro Gly Gly Gly Phe
Leu Val Gly Leu Ile Asp Phe Val 50 55
60 tgg gga ata gtt ggc cct tct caa tgg gac
gca ttt cta gta caa att 240Trp Gly Ile Val Gly Pro Ser Gln Trp Asp
Ala Phe Leu Val Gln Ile 65 70 75
80 gaa caa tta att aat gaa aga ata gct gaa ttt gct
agg aac gct gct 288Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala
Arg Asn Ala Ala 85 90
95 att gct aat tta gaa gga tta gga aac aat ttc aac atc tat
gtg gaa 336Ile Ala Asn Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr
Val Glu 100 105 110
gca ttt aag gaa tgg gaa gaa gat cct aag aat cca gca acg agg acc
384Ala Phe Lys Glu Trp Glu Glu Asp Pro Lys Asn Pro Ala Thr Arg Thr
115 120 125 aga
gta att gat cgc ttt cgt ata ctt gat ggg cta ctt gaa agg gac 432Arg
Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu Glu Arg Asp 130
135 140 att cct tcg
ttt cga att tct gga ttt gaa gta ccc ctt ctc tcc gtt 480Ile Pro Ser
Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145
150 155 160 tat gct caa gcg gcc
aat ctg cat cta gct atc tta aga gat tct gtc 528Tyr Ala Gln Ala Ala
Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165
170 175 atc ttt gga gaa aga tgg gga
ttg aca acg ata aat gtc aat gaa aac 576Ile Phe Gly Glu Arg Trp Gly
Leu Thr Thr Ile Asn Val Asn Glu Asn 180
185 190 tat aat aga cta att agg cat att gat
gaa tat gct gat cac tgt gca 624Tyr Asn Arg Leu Ile Arg His Ile Asp
Glu Tyr Ala Asp His Cys Ala 195 200
205 aat acg tat aat cgg gga tta aat aat tta ccg
aaa tct acg tat caa 672Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro
Lys Ser Thr Tyr Gln 210 215 220
gat tgg ata aca tat aat cga ttg cgg aga gac tta aca
ttg act gta 720Asp Trp Ile Thr Tyr Asn Arg Leu Arg Arg Asp Leu Thr
Leu Thr Val 225 230 235
240 tta gat atc gcc gct ttc ttt cca aac tat gac aat agg aga tat
cca 768Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr Asp Asn Arg Arg Tyr
Pro 245 250 255
att cag cca gtt ggt caa cta aca agg gaa gtt tat acg gac cca tta
816Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro Leu
260 265 270 att
aat ttt aat cca cag tta cag tct gta gct caa tta cct act ttt 864Ile
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe
275 280 285 aac gtt atg
gag aac agc gca att aga aat cct cat ttg ttc gac ata 912Asn Val Met
Glu Asn Ser Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290
295 300 ttg aat aat ctt aca
atc ttt acg gat tgg ttt agt gtt gga cgc aac 960Leu Asn Asn Leu Thr
Ile Phe Thr Asp Trp Phe Ser Val Gly Arg Asn 305 310
315 320 ttc tat tgg gga gga cat cga
gta ata tct agc ctt ata gga ggt ggg 1008Phe Tyr Trp Gly Gly His Arg
Val Ile Ser Ser Leu Ile Gly Gly Gly 325
330 335 aac atc aca tcg cct atc tat gga aga gag
gcg aac caa gag cct cca 1056Asn Ile Thr Ser Pro Ile Tyr Gly Arg Glu
Ala Asn Gln Glu Pro Pro 340 345
350 aga tcc ttt act ttt aat gga ccc gtg ttt agg act tta
tca aat cct 1104Arg Ser Phe Thr Phe Asn Gly Pro Val Phe Arg Thr Leu
Ser Asn Pro 355 360 365
act tta cga tta tta cag caa cct tgg cca gcg cca cca ttt aat tta
1152Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro Pro Phe Asn Leu
370 375 380 cgt
ggt gtt gaa gga gta gaa ttt tct aca cct aca aat agc ttt acg 1200Arg
Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385
390 395 400 tat cga gga aga
ggg acg gtt gat tct tta act gaa ttg ccg cct gag 1248Tyr Arg Gly Arg
Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405
410 415 gat aat agt gtg cca cct cgc
gaa gga tat agt cat cgt tta tgt cat 1296Asp Asn Ser Val Pro Pro Arg
Glu Gly Tyr Ser His Arg Leu Cys His 420
425 430 gca acc ttt gtt caa aga tcg gga aca
cct ttc tta aca act ggt gta 1344Ala Thr Phe Val Gln Arg Ser Gly Thr
Pro Phe Leu Thr Thr Gly Val 435 440
445 gta ttc tct tgg acg cat cgt agt gca act ctt aca
aat aca atc gac 1392Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr
Asn Thr Ile Asp 450 455 460
cca gag aga att aat caa ata cct tta gtg aag gga ttt aga gtt
tgg 1440Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val
Trp 465 470 475 480
ggg ggc acc tct gtc att acc gga ccc gga ttt acc gga ggg gat atc
1488Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile
485 490 495 ctt cga
aga aat acc ttt ggt gat ttc gta tct cta caa gtc aac att 1536Leu Arg
Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile
500 505 510 aat tca cca att
acc caa aga tac cgt tta aga ttt cgt tac gct tcc 1584Asn Ser Pro Ile
Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 515
520 525 agt agg gat gca cga gtt ata
gta tta acg gga gcg gca tcc acc gga 1632Ser Arg Asp Ala Arg Val Ile
Val Leu Thr Gly Ala Ala Ser Thr Gly 530 535
540 gtg gga ggc caa gtt agt gta aat atg cct
ctt cag aaa act atg gaa 1680Val Gly Gly Gln Val Ser Val Asn Met Pro
Leu Gln Lys Thr Met Glu 545 550 555
560 ata ggg gag aac tta aca tcc aga aca ttt aga tat acc
gat ttt agt 1728Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr
Asp Phe Ser 565 570
575 aat cct ttt tca ttt aga gct aat cca gat ata att ggg ata agt
gaa 1776Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser
Glu 580 585 590
caa cct cta ttt ggg gcg ggt tct att agt agc ggt gaa ctt tac ata
1824Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile
595 600 605 gat aaa
att gaa att att cta gca gat gca aca ttt gaa tga 1866Asp Lys
Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu 610
615 620 30621PRTArtificial
SequenceSynthetic Construct 30Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile
Pro Tyr Asn Cys Leu 1 5 10
15 Ser Asn Pro Glu Glu Val Leu Leu Asp Gly Glu Arg Ile Ser Thr Gly
20 25 30 Asn Ser
Ser Ile Asp Ile Ser Leu Ser Leu Val Gln Phe Leu Val Ser 35
40 45 Asn Phe Val Pro Gly Gly Gly
Phe Leu Val Gly Leu Ile Asp Phe Val 50 55
60 Trp Gly Ile Val Gly Pro Ser Gln Trp Asp Ala Phe
Leu Val Gln Ile 65 70 75
80 Glu Gln Leu Ile Asn Glu Arg Ile Ala Glu Phe Ala Arg Asn Ala Ala
85 90 95 Ile Ala Asn
Leu Glu Gly Leu Gly Asn Asn Phe Asn Ile Tyr Val Glu 100
105 110 Ala Phe Lys Glu Trp Glu Glu Asp
Pro Lys Asn Pro Ala Thr Arg Thr 115 120
125 Arg Val Ile Asp Arg Phe Arg Ile Leu Asp Gly Leu Leu
Glu Arg Asp 130 135 140
Ile Pro Ser Phe Arg Ile Ser Gly Phe Glu Val Pro Leu Leu Ser Val 145
150 155 160 Tyr Ala Gln Ala
Ala Asn Leu His Leu Ala Ile Leu Arg Asp Ser Val 165
170 175 Ile Phe Gly Glu Arg Trp Gly Leu Thr
Thr Ile Asn Val Asn Glu Asn 180 185
190 Tyr Asn Arg Leu Ile Arg His Ile Asp Glu Tyr Ala Asp His
Cys Ala 195 200 205
Asn Thr Tyr Asn Arg Gly Leu Asn Asn Leu Pro Lys Ser Thr Tyr Gln 210
215 220 Asp Trp Ile Thr Tyr
Asn Arg Leu Arg Arg Asp Leu Thr Leu Thr Val 225 230
235 240 Leu Asp Ile Ala Ala Phe Phe Pro Asn Tyr
Asp Asn Arg Arg Tyr Pro 245 250
255 Ile Gln Pro Val Gly Gln Leu Thr Arg Glu Val Tyr Thr Asp Pro
Leu 260 265 270 Ile
Asn Phe Asn Pro Gln Leu Gln Ser Val Ala Gln Leu Pro Thr Phe 275
280 285 Asn Val Met Glu Asn Ser
Ala Ile Arg Asn Pro His Leu Phe Asp Ile 290 295
300 Leu Asn Asn Leu Thr Ile Phe Thr Asp Trp Phe
Ser Val Gly Arg Asn 305 310 315
320 Phe Tyr Trp Gly Gly His Arg Val Ile Ser Ser Leu Ile Gly Gly Gly
325 330 335 Asn Ile
Thr Ser Pro Ile Tyr Gly Arg Glu Ala Asn Gln Glu Pro Pro 340
345 350 Arg Ser Phe Thr Phe Asn Gly
Pro Val Phe Arg Thr Leu Ser Asn Pro 355 360
365 Thr Leu Arg Leu Leu Gln Gln Pro Trp Pro Ala Pro
Pro Phe Asn Leu 370 375 380
Arg Gly Val Glu Gly Val Glu Phe Ser Thr Pro Thr Asn Ser Phe Thr 385
390 395 400 Tyr Arg Gly
Arg Gly Thr Val Asp Ser Leu Thr Glu Leu Pro Pro Glu 405
410 415 Asp Asn Ser Val Pro Pro Arg Glu
Gly Tyr Ser His Arg Leu Cys His 420 425
430 Ala Thr Phe Val Gln Arg Ser Gly Thr Pro Phe Leu Thr
Thr Gly Val 435 440 445
Val Phe Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 450
455 460 Pro Glu Arg Ile
Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 465 470
475 480 Gly Gly Thr Ser Val Ile Thr Gly Pro
Gly Phe Thr Gly Gly Asp Ile 485 490
495 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val
Asn Ile 500 505 510
Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser
515 520 525 Ser Arg Asp Ala
Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 530
535 540 Val Gly Gly Gln Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu 545 550
555 560 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr
Thr Asp Phe Ser 565 570
575 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu
580 585 590 Gln Pro Leu
Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 595
600 605 Asp Lys Ile Glu Ile Ile Leu Ala
Asp Ala Thr Phe Glu 610 615 620
311428DNABacillus thuringiensisCDS(1)..(1428)Native DNA sequence encoding
Bacillus thuringiensis Cry6Aa toxin 31atg att att gat agt aaa acg
act tta cct aga cat tca ctt att cat 48Met Ile Ile Asp Ser Lys Thr
Thr Leu Pro Arg His Ser Leu Ile His 1 5
10 15 aca att aaa tta aat tct aat aag aaa
tat ggt cct ggt gat atg act 96Thr Ile Lys Leu Asn Ser Asn Lys Lys
Tyr Gly Pro Gly Asp Met Thr 20 25
30 aat gga aat caa ttt att att tca aaa caa gaa
tgg gct acg att gga 144Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu
Trp Ala Thr Ile Gly 35 40
45 gca tat att cag act gga tta ggt tta cca gta aat gaa
caa caa tta 192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu
Gln Gln Leu 50 55 60
aga aca cat gtt aat tta agt cag gat ata tca ata cct agt gat
ttt 240Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp
Phe 65 70 75 80
tct caa tta tat gat gtt tat tgt tct gat aaa act tca gca gaa tgg
288Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp
85 90 95 tgg
aat aaa aat tta tat cct tta att att aaa tct gct aat gat att 336Trp
Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile
100 105 110 gct tca tat
ggt ttt aaa gtt gct ggt gat cct tct att aag aaa gat 384Ala Ser Tyr
Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115
120 125 gga tat ttt aaa aaa
ttg caa gat gaa tta gat aat att gtt gat aat 432Gly Tyr Phe Lys Lys
Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130
135 140 aat tcc gat gat gat gca ata
gct aaa gct att aaa gat ttt aaa gcg 480Asn Ser Asp Asp Asp Ala Ile
Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150
155 160 cga tgt ggt att tta att aaa gaa gct
aaa caa tat gaa gaa gct gca 528Arg Cys Gly Ile Leu Ile Lys Glu Ala
Lys Gln Tyr Glu Glu Ala Ala 165 170
175 aaa aat att gta aca tct tta gat caa ttt tta
cat ggt gat cag aaa 576Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu
His Gly Asp Gln Lys 180 185
190 aaa tta gaa ggt gtt atc aat att caa aaa cgt tta aaa
gaa gtt caa 624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys
Glu Val Gln 195 200 205
aca gct ctt aat caa gcc cat ggg gaa agt agt cca gct cat aaa
gag 672Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys
Glu 210 215 220
tta tta gaa aaa gta aaa aat tta aaa aca aca tta gaa agg act att
720Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile
225 230 235 240 aaa
gct gaa caa gat tta gag aaa aaa gta gaa tat agt ttt cta tta 768Lys
Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu
245 250 255 gga cca ttg
tta gga ttt gtt gtt tat gaa att ctt gaa aat act gct 816Gly Pro Leu
Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260
265 270 gtt cag cat ata aaa
aat caa att gat gag ata aag aaa caa tta gat 864Val Gln His Ile Lys
Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275
280 285 tct gct cag cat gat ttg gat
aga gat gtt aaa att ata gga atg tta 912Ser Ala Gln His Asp Leu Asp
Arg Asp Val Lys Ile Ile Gly Met Leu 290 295
300 aat agt att aat aca gat att gat aat
tta tat agt caa gga caa gaa 960Asn Ser Ile Asn Thr Asp Ile Asp Asn
Leu Tyr Ser Gln Gly Gln Glu 305 310
315 320 gca att aaa gtt ttc caa aag tta caa ggt att
tgg gct act att gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile
Trp Ala Thr Ile Gly 325 330
335 gct caa ata gaa aat ctt aga aca acg tcg tta caa gaa gtt
caa gat 1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val
Gln Asp 340 345 350
tct gat gat gct gat gag ata caa att gaa ctt gag gac gct tct gat
1104Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp
355 360 365 gct tgg
tta gtt gtg gct caa gaa gct cgt gat ttt aca cta aat gct 1152Ala Trp
Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370
375 380 tat tca act aat agt
aga caa aat tta ccg att aat gtt ata tca gat 1200Tyr Ser Thr Asn Ser
Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390
395 400 tca tgt aat tgt tca aca aca aat
atg aca tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser Thr Thr Asn
Met Thr Ser Asn Gln Tyr Ser Asn 405
410 415 cca aca aca aat atg aca tca aat caa tat
atg att tca cat gaa tat 1296Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr
Met Ile Ser His Glu Tyr 420 425
430 aca agt tta cca aat aat ttt atg tta tca aga aat agt
aat tta gaa 1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser
Asn Leu Glu 435 440 445
tat aaa tgt cct gaa aat aat ttt atg ata tat tgg tat aat aat tcg
1392Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser
450 455 460 gat
tgg tat aat aat tcg gat tgg tat aat aat tga 1428Asp
Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465
470 475
32475PRTBacillus thuringiensis 32Met Ile Ile Asp Ser Lys Thr Thr Leu Pro
Arg His Ser Leu Ile His 1 5 10
15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met
Thr 20 25 30 Asn
Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35
40 45 Ala Tyr Ile Gln Thr Gly
Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55
60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser
Ile Pro Ser Asp Phe 65 70 75
80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp
85 90 95 Trp Asn
Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100
105 110 Ala Ser Tyr Gly Phe Lys Val
Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120
125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn
Ile Val Asp Asn 130 135 140
Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145
150 155 160 Arg Cys Gly
Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165
170 175 Lys Asn Ile Val Thr Ser Leu Asp
Gln Phe Leu His Gly Asp Gln Lys 180 185
190 Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys
Glu Val Gln 195 200 205
Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210
215 220 Leu Leu Glu Lys
Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230
235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys
Val Glu Tyr Ser Phe Leu Leu 245 250
255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn
Thr Ala 260 265 270
Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp
275 280 285 Ser Ala Gln His
Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290
295 300 Asn Ser Ile Asn Thr Asp Ile Asp
Asn Leu Tyr Ser Gln Gly Gln Glu 305 310
315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp
Ala Thr Ile Gly 325 330
335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp
340 345 350 Ser Asp Asp
Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355
360 365 Ala Trp Leu Val Val Ala Gln Glu
Ala Arg Asp Phe Thr Leu Asn Ala 370 375
380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val
Ile Ser Asp 385 390 395
400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn
405 410 415 Pro Thr Thr Asn
Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420
425 430 Thr Ser Leu Pro Asn Asn Phe Met Leu
Ser Arg Asn Ser Asn Leu Glu 435 440
445 Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn
Asn Ser 450 455 460
Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470
475 331428DNAArtificial SequenceSynthetic DNA sequence encoding
Bacillus thuringiensis Cry6Aa toxin using codons optimized for maize
and Table 1 sequences are maintainedCDS(1)..(1428) 33atg atc atc gac
tcc aag acg acc ctg cca cgg cac tcc ctt atc cac 48Met Ile Ile Asp
Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5
10 15 aca att aaa tta aat agc
aat aag aag tac ggt ccc ggt gat atg act 96Thr Ile Lys Leu Asn Ser
Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20
25 30 aac gga aat caa ttc att att tca
aag caa gag tgg gct acc atc gga 144Asn Gly Asn Gln Phe Ile Ile Ser
Lys Gln Glu Trp Ala Thr Ile Gly 35 40
45 gcg tac atc cag act ggg ctg ggc cta cca
gta aat gaa caa caa tta 192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro
Val Asn Glu Gln Gln Leu 50 55
60 agg acc cat gtc aac ctc agc caa gat atc agc atc
cct agc gac ttt 240Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile
Pro Ser Asp Phe 65 70 75
80 tct cag ctc tac gac gtc tat tgc agc gat aaa act tcc gca
gaa tgg 288Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala
Glu Trp 85 90 95
tgg aat aaa aac ctg tac ccc ctc atc att aaa tct gcc aac gat att
336Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile
100 105 110 gcc
agc tac ggc ttc aag gtc gcg ggt gat cct tct att aag aag gac 384Ala
Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp
115 120 125 ggc tac ttc
aag aag ctg caa gat gag ctg gac aac att gtt gac aat 432Gly Tyr Phe
Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile Val Asp Asn 130
135 140 aat tcc gat gat gat
gca ata gcg aaa gcc att aaa gac ttc aag gcg 480Asn Ser Asp Asp Asp
Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145 150
155 160 cga tgc ggc atc cta att aaa
gaa gca aag cag tat gaa gag gca gcg 528Arg Cys Gly Ile Leu Ile Lys
Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165
170 175 aaa aat atc gta aca tcc ctc gac caa
ttt ctg cat ggc gat cag aag 576Lys Asn Ile Val Thr Ser Leu Asp Gln
Phe Leu His Gly Asp Gln Lys 180 185
190 aaa ttg gag ggt gtg atc aac atc caa aaa cgt
ctg aag gag gtg cag 624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg
Leu Lys Glu Val Gln 195 200
205 acg gct ctt aat caa gcc cac ggg gaa agt tca cca gct
cat aaa gag 672Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala
His Lys Glu 210 215 220
ctg tta gag aaa gtc aag aat ctc aag acc aca ctt gag agg acc
att 720Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr
Ile 225 230 235 240
aaa gct gag caa gac ctg gag aag aaa gtg gag tac agt ttc ctt ctc
768Lys Ala Glu Gln Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu
245 250 255 ggc
ccc ttg ctg ggc ttc gtc gtt tat gaa atc ctt gaa aat act gcc 816Gly
Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala
260 265 270 gtc cag cat
ata aaa aac caa att gac gag ata aag aag caa ctg gac 864Val Gln His
Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275
280 285 tct gcc cag cac gac
ttg gac aga gac gtt aag atc ata ggg atg ctg 912Ser Ala Gln His Asp
Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290
295 300 aac agt att aat aca gac att
gat aac ttg tat agc caa gga caa gag 960Asn Ser Ile Asn Thr Asp Ile
Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310
315 320 gca att aaa gtg ttc caa aag ctc caa
ggc atc tgg gca act atc gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln
Gly Ile Trp Ala Thr Ile Gly 325 330
335 gcg cag ata gag aac ctt agg aca acg tcg ctc caa
gaa gtg caa gac 1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln
Glu Val Gln Asp 340 345
350 tct gac gac gcc gat gag atc caa att gaa ctt gag gac gcg
tct gat 1104Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala
Ser Asp 355 360 365
gct tgg tta gtg gtg gcc caa gaa gct cgc gac ttc aca cta aat gcc
1152Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala
370 375 380 tac tca
act aac tcg cgt cag aat cta ccg att aat gtt ata tcc gat 1200Tyr Ser
Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385
390 395 400 tcc tgc aac tgt tcc
aca acg aac atg acc tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser
Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405
410 415 cca acc aca aat atg acc tca aat
caa tat atg atc tca cac gag tat 1296Pro Thr Thr Asn Met Thr Ser Asn
Gln Tyr Met Ile Ser His Glu Tyr 420 425
430 acc tcg ttg ccg aat aat ttc atg ctc tca aga
aat agc aat ctg gaa 1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg
Asn Ser Asn Leu Glu 435 440
445 tat aag tgt cct gaa aat aat ttc atg ata tac tgg tac
aat aat tcg 1392Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr
Asn Asn Ser 450 455 460
gac tgg tac aat aat tcg gat tgg tac aat aat tga
1428Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn
465 470 475
34475PRTArtificial SequenceSynthetic Construct 34Met Ile Ile Asp Ser Lys
Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5
10 15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly
Pro Gly Asp Met Thr 20 25
30 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile
Gly 35 40 45 Ala
Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50
55 60 Arg Thr His Val Asn Leu
Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 70
75 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys
Thr Ser Ala Glu Trp 85 90
95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile
100 105 110 Ala Ser
Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys Lys Asp 115
120 125 Gly Tyr Phe Lys Lys Leu Gln
Asp Glu Leu Asp Asn Ile Val Asp Asn 130 135
140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys
Asp Phe Lys Ala 145 150 155
160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala
165 170 175 Lys Asn Ile
Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln Lys 180
185 190 Lys Leu Glu Gly Val Ile Asn Ile
Gln Lys Arg Leu Lys Glu Val Gln 195 200
205 Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala
His Lys Glu 210 215 220
Leu Leu Glu Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225
230 235 240 Lys Ala Glu Gln
Asp Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245
250 255 Gly Pro Leu Leu Gly Phe Val Val Tyr
Glu Ile Leu Glu Asn Thr Ala 260 265
270 Val Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln
Leu Asp 275 280 285
Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290
295 300 Asn Ser Ile Asn Thr
Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310
315 320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly
Ile Trp Ala Thr Ile Gly 325 330
335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln
Asp 340 345 350 Ser
Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355
360 365 Ala Trp Leu Val Val Ala
Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370 375
380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile
Asn Val Ile Ser Asp 385 390 395
400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn
405 410 415 Pro Thr
Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser His Glu Tyr 420
425 430 Thr Ser Leu Pro Asn Asn Phe
Met Leu Ser Arg Asn Ser Asn Leu Glu 435 440
445 Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp
Tyr Asn Asn Ser 450 455 460
Asp Trp Tyr Asn Asn Ser Asp Trp Tyr Asn Asn 465 470
475 351428DNAArtificial SequenceSynthetic DNA sequence in
accordance with the invention encoding Bacillus thuringiensis Cry6Aa
toxin using codons optimized for maize and with sequences identified
in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(1428) 35atg atc atc gac tcc aag acg acc ctg cca cgg
cac tcc ctt atc cac 48Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg
His Ser Leu Ile His 1 5 10
15 aca att aaa tta aat agc aat aag aag tac ggt ccc ggt
gat atg act 96Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly
Asp Met Thr 20 25 30
aac gga aat caa ttc att atc tca aag caa gag tgg gct acc atc
gga 144Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile
Gly 35 40 45
gcg tac atc cag act ggg ctg ggc cta cca gta aat gaa caa caa tta
192Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu
50 55 60 agg
acc cat gtc aac ctc agc caa gat atc agc atc cct agc gac ttt 240Arg
Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65
70 75 80 tct cag ctc
tac gac gtc tat tgc agc gat aaa act tcc gca gaa tgg 288Ser Gln Leu
Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp
85 90 95 tgg aat aaa aac ctg
tac ccc ctc atc att aaa tct gcc aac gat att 336Trp Asn Lys Asn Leu
Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100
105 110 gcc agc tac ggc ttc aag gtc
gcg ggt gat cct tct att aag aag gac 384Ala Ser Tyr Gly Phe Lys Val
Ala Gly Asp Pro Ser Ile Lys Lys Asp 115 120
125 ggc tac ttc aag aag ctg caa gat gag
ctg gac aac att gtt gac aat 432Gly Tyr Phe Lys Lys Leu Gln Asp Glu
Leu Asp Asn Ile Val Asp Asn 130 135
140 aat tcc gat gat gat gca ata gcg aaa gcc att
aaa gac ttc aag gcg 480Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile
Lys Asp Phe Lys Ala 145 150 155
160 cga tgc ggc atc cta att aaa gaa gca aag cag tat gaa
gag gca gcg 528Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys Gln Tyr Glu
Glu Ala Ala 165 170
175 aaa aat atc gta aca tcc ctc gac caa ttt ctg cat ggc gat cag
aag 576Lys Asn Ile Val Thr Ser Leu Asp Gln Phe Leu His Gly Asp Gln
Lys 180 185 190
aaa ttg gag ggt gtg atc aac atc caa aaa cgt ctg aag gag gtg cag
624Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu Val Gln
195 200 205 acg
gct ctt aat caa gcc cac ggg gaa agt tca cca gct cat aaa gag 672Thr
Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210
215 220 ctg tta gag
aaa gtc aag aat ctc aag acc aca ctt gag agg acc att 720Leu Leu Glu
Lys Val Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225
230 235 240 aaa gct gag caa gac
ctg gag aag aaa gtg gag tac agt ttc ctt ctc 768Lys Ala Glu Gln Asp
Leu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245
250 255 ggc ccc ttg ctg ggc ttc gtc
gtt tat gaa atc ctt gaa aat act gcc 816Gly Pro Leu Leu Gly Phe Val
Val Tyr Glu Ile Leu Glu Asn Thr Ala 260
265 270 gtc cag cat ata aaa aac caa att gac
gag ata aag aag caa ctg gac 864Val Gln His Ile Lys Asn Gln Ile Asp
Glu Ile Lys Lys Gln Leu Asp 275 280
285 tct gcc cag cac gac ttg gac aga gac gtt aag
atc ata ggg atg ctg 912Ser Ala Gln His Asp Leu Asp Arg Asp Val Lys
Ile Ile Gly Met Leu 290 295 300
aac agt att aat aca gac att gat aac ttg tat agc caa
gga caa gag 960Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln
Gly Gln Glu 305 310 315
320 gca att aaa gtg ttc caa aag ctc caa ggc atc tgg gca act atc
gga 1008Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile
Gly 325 330 335
gcg cag ata gag aac ctt agg aca acg tcg ctc caa gaa gtg caa gac
1056Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp
340 345 350 tct gac
gac gcc gat gag atc caa att gaa ctt gag gac gcg tct gat 1104Ser Asp
Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala Ser Asp 355
360 365 gct tgg tta gtg gtg
gcc caa gaa gct cgc gac ttc aca cta aat gcc 1152Ala Trp Leu Val Val
Ala Gln Glu Ala Arg Asp Phe Thr Leu Asn Ala 370
375 380 tac tca act aac tcg cgt cag
aat cta ccg att aat gtt atc tcc gat 1200Tyr Ser Thr Asn Ser Arg Gln
Asn Leu Pro Ile Asn Val Ile Ser Asp 385 390
395 400 tcc tgc aac tgt tcc aca acg aac atg acc
tca aat caa tac agt aat 1248Ser Cys Asn Cys Ser Thr Thr Asn Met Thr
Ser Asn Gln Tyr Ser Asn 405 410
415 cca acc aca aat atg acc tca aat caa tac atg atc tca
cac gag tat 1296Pro Thr Thr Asn Met Thr Ser Asn Gln Tyr Met Ile Ser
His Glu Tyr 420 425 430
acc tcg ttg ccg aat aat ttc atg ctc tca aga aat agc aat ctg gaa
1344Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser Asn Leu Glu
435 440 445 tat
aag tgt cct gaa aat aat ttc atg ata tac tgg tac aat aat tcg 1392Tyr
Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450
455 460 gac tgg tac aat
aat tcg gat tgg tac aat aat tga 1428Asp Trp Tyr Asn
Asn Ser Asp Trp Tyr Asn Asn 465
470 475 36475PRTArtificial
SequenceSynthetic Construct 36Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg
His Ser Leu Ile His 1 5 10
15 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr
20 25 30 Asn Gly
Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35
40 45 Ala Tyr Ile Gln Thr Gly Leu
Gly Leu Pro Val Asn Glu Gln Gln Leu 50 55
60 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile
Pro Ser Asp Phe 65 70 75
80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp
85 90 95 Trp Asn Lys
Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile 100
105 110 Ala Ser Tyr Gly Phe Lys Val Ala
Gly Asp Pro Ser Ile Lys Lys Asp 115 120
125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile
Val Asp Asn 130 135 140
Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys Asp Phe Lys Ala 145
150 155 160 Arg Cys Gly Ile
Leu Ile Lys Glu Ala Lys Gln Tyr Glu Glu Ala Ala 165
170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln
Phe Leu His Gly Asp Gln Lys 180 185
190 Lys Leu Glu Gly Val Ile Asn Ile Gln Lys Arg Leu Lys Glu
Val Gln 195 200 205
Thr Ala Leu Asn Gln Ala His Gly Glu Ser Ser Pro Ala His Lys Glu 210
215 220 Leu Leu Glu Lys Val
Lys Asn Leu Lys Thr Thr Leu Glu Arg Thr Ile 225 230
235 240 Lys Ala Glu Gln Asp Leu Glu Lys Lys Val
Glu Tyr Ser Phe Leu Leu 245 250
255 Gly Pro Leu Leu Gly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr
Ala 260 265 270 Val
Gln His Ile Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275
280 285 Ser Ala Gln His Asp Leu
Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295
300 Asn Ser Ile Asn Thr Asp Ile Asp Asn Leu Tyr
Ser Gln Gly Gln Glu 305 310 315
320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly
325 330 335 Ala Gln
Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp 340
345 350 Ser Asp Asp Ala Asp Glu Ile
Gln Ile Glu Leu Glu Asp Ala Ser Asp 355 360
365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe
Thr Leu Asn Ala 370 375 380
Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn Val Ile Ser Asp 385
390 395 400 Ser Cys Asn
Cys Ser Thr Thr Asn Met Thr Ser Asn Gln Tyr Ser Asn 405
410 415 Pro Thr Thr Asn Met Thr Ser Asn
Gln Tyr Met Ile Ser His Glu Tyr 420 425
430 Thr Ser Leu Pro Asn Asn Phe Met Leu Ser Arg Asn Ser
Asn Leu Glu 435 440 445
Tyr Lys Cys Pro Glu Asn Asn Phe Met Ile Tyr Trp Tyr Asn Asn Ser 450
455 460 Asp Trp Tyr Asn
Asn Ser Asp Trp Tyr Asn Asn 465 470 475
37888DNASphingobiurn herbicidovoransCDS(1)..(888)Native DNA sequence
encoding Sphingobiurn herbicidovorans AAD1 protein 37atg cat gct gca
ctg tcc ccc ctc tcc cag cgc ttt gag cgc atc gcg 48Met His Ala Ala
Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5
10 15 gtc cag ccg ctg acc ggc
gtc ctg ggc gcc gag atc acc ggc gtc gac 96Val Gln Pro Leu Thr Gly
Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20
25 30 ctg cgc gag ccg ctc gac gac agc
acc tgg aac gaa atc ctc gac gcg 144Leu Arg Glu Pro Leu Asp Asp Ser
Thr Trp Asn Glu Ile Leu Asp Ala 35 40
45 ttc cac act tac cag gtc atc tat ttt ccc
ggc cag gcg atc acc aac 192Phe His Thr Tyr Gln Val Ile Tyr Phe Pro
Gly Gln Ala Ile Thr Asn 50 55
60 gaa cag cac atc gcc ttc agc cgg cgc ttc ggc ccc
gtc gat ccc gtg 240Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro
Val Asp Pro Val 65 70 75
80 ccc ctg ctc aag agc atc gaa ggg tat cca gag gtg cag atg
atc cgc 288Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met
Ile Arg 85 90 95
cgc gaa gcc aac gaa agc ggg cgt gtg atc ggt gat gac tgg cac acc
336Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr
100 105 110 gac
agc acc ttc ctg gac gca ccg ccg gcc gcc gtg gtg atg cgc gcg 384Asp
Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala
115 120 125 atc gac gtg
ccc gag cat ggc ggc gac acc ggt ttt ctg agc atg tac 432Ile Asp Val
Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130
135 140 acc gcg tgg gag acg
ctg tcg ccc acc atg cag gcc acc atc gaa ggg 480Thr Ala Trp Glu Thr
Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145 150
155 160 ttg aac gta gtg cac agc gcc
acg cgt gtg ttc ggc tcg ctc tac cag 528Leu Asn Val Val His Ser Ala
Thr Arg Val Phe Gly Ser Leu Tyr Gln 165
170 175 gcc cag aac cgg cgc ttc agc aac acc
agc gtc aag gtg atg gac gtc 576Ala Gln Asn Arg Arg Phe Ser Asn Thr
Ser Val Lys Val Met Asp Val 180 185
190 gac gcg ggc gac cgt gaa acc gtg cac ccc ctg
gtg gtg acc cat ccg 624Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu
Val Val Thr His Pro 195 200
205 ggc agc ggc cgc aag ggc ctg tac gtg aac cag gtc tat
tgc cag cgc 672Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr
Cys Gln Arg 210 215 220
atc gag ggc atg acc gat gcc gaa agc aaa ccg ctg ctg cag ttc
ctg 720Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe
Leu 225 230 235 240
tac gag cat gcg aca cgg ttc gat ttc acc tgc cgc gtg cgc tgg aag
768Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys
245 250 255 aag
gac cag gtc ctg gtc tgg gac aac ctg tgc acg atg cac cgg gcc 816Lys
Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala
260 265 270 gta ccc gac
tac gcg ggc aag ttc cgc tac ctg acg cgc acc acg gtc 864Val Pro Asp
Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275
280 285 ggt ggc gtg cgc ccg
gcg cgc tag 888Gly Gly Val Arg Pro
Ala Arg 290
295 38295PRTSphingobiurn
herbicidovorans 38Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg
Ile Ala 1 5 10 15
Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp
20 25 30 Leu Arg Glu Pro Leu
Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35
40 45 Phe His Thr Tyr Gln Val Ile Tyr Phe
Pro Gly Gln Ala Ile Thr Asn 50 55
60 Glu Gln His Ile Ala Phe Ser Arg Arg Phe Gly Pro Val
Asp Pro Val 65 70 75
80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg
85 90 95 Arg Glu Ala Asn
Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr 100
105 110 Asp Ser Thr Phe Leu Asp Ala Pro Pro
Ala Ala Val Val Met Arg Ala 115 120
125 Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser
Met Tyr 130 135 140
Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145
150 155 160 Leu Asn Val Val His
Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln 165
170 175 Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser
Val Lys Val Met Asp Val 180 185
190 Asp Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His
Pro 195 200 205 Gly
Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210
215 220 Ile Glu Gly Met Thr Asp
Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230
235 240 Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys
Arg Val Arg Trp Lys 245 250
255 Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg Ala
260 265 270 Val Pro
Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val 275
280 285 Gly Gly Val Arg Pro Ala Arg
290 295 39888DNAArtificial SequenceSynthetic DNA
sequence encoding the AAD1 protein using codons optimized for maize
and Table 1 and Table 2 sequences are maintainedCDS(1)..(888) 39atg
cac gct gca ctg tca cca ctc tca cag cgc ttt gag aga att gcg 48Met
His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1
5 10 15 gtc cag ccg
ctg act ggc gtc ttg ggc gct gag atc acc ggc gtc gat 96Val Gln Pro
Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly Val Asp 20
25 30 ctg agg gag cct ctc
gac gat tca acg tgg aac gaa att ctc gac gcg 144Leu Arg Glu Pro Leu
Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala 35
40 45 ttc cat act tac caa gtc atc
tat ttt ccc ggg caa gct att acc aac 192Phe His Thr Tyr Gln Val Ile
Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50 55
60 gaa caa cac atc gct ttc tct cgg cga
ttc ggc ccc gtc gat cca gtg 240Glu Gln His Ile Ala Phe Ser Arg Arg
Phe Gly Pro Val Asp Pro Val 65 70
75 80 ccc tta ctc aag tct atc gaa ggc tac cca gag
gtg cag atg ata aga 288Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu
Val Gln Met Ile Arg 85 90
95 agg gag gcc aac gaa agc ggg cgt gtg ata ggt gat gac
tgg cac act 336Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp
Trp His Thr 100 105 110
gac agc aca ttc ctg gat gca ccg ccg gcc gct gtg gtg atg agg
gca 384Asp Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg
Ala 115 120 125
atc gac gtg ccc gag cac gga ggt gac act ggt ttc ttg agt atg tac
432Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr
130 135 140 act
gct tgg gag acg ctt tcg cct act atg caa gcc aca atc gag ggg 480Thr
Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr Ile Glu Gly 145
150 155 160 ttg aat gta
gtt cac agc gcc acg cgt gtg ttc gga tct ctc tat caa 528Leu Asn Val
Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln
165 170 175 gcc caa aac cgg cgc
ttt tca aat acc tcc gtc aag gtg atg gac gtt 576Ala Gln Asn Arg Arg
Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180
185 190 gac gcg ggc gac cgt gaa acc
gtg cac cct ctt gtt gta acc cat ccg 624Asp Ala Gly Asp Arg Glu Thr
Val His Pro Leu Val Val Thr His Pro 195 200
205 ggc agt ggt cgc aag ggc cta tac gtt
aac caa gtc tat tgc cag cgc 672Gly Ser Gly Arg Lys Gly Leu Tyr Val
Asn Gln Val Tyr Cys Gln Arg 210 215
220 atc gag gga atg aca gac gca gag agt aag ccg
ctc ctg caa ttc ctg 720Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro
Leu Leu Gln Phe Leu 225 230 235
240 tac gag cac gcg aca cgg ttc gat ttc acc tgc cgc gtg
cgc tgg aaa 768Tyr Glu His Ala Thr Arg Phe Asp Phe Thr Cys Arg Val
Arg Trp Lys 245 250
255 aag gat caa gtc ctt gta tgg gac aac ctt tgt acg atg cac cgg
gcc 816Lys Asp Gln Val Leu Val Trp Asp Asn Leu Cys Thr Met His Arg
Ala 260 265 270
gtt cct gac tac gcg ggc aag ttc aga tac ctg acg agg acc acg gtc
864Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr Thr Val
275 280 285 ggt
gga gtt agg cca gcg aga tga 888Gly
Gly Val Arg Pro Ala Arg 290
295
40295PRTArtificial SequenceSynthetic Construct 40Met His Ala Ala Leu Ser
Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5
10 15 Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu
Ile Thr Gly Val Asp 20 25
30 Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp
Ala 35 40 45 Phe
His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50
55 60 Glu Gln His Ile Ala Phe
Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70
75 80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro Glu
Val Gln Met Ile Arg 85 90
95 Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His Thr
100 105 110 Asp Ser
Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115
120 125 Ile Asp Val Pro Glu His Gly
Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135
140 Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala
Thr Ile Glu Gly 145 150 155
160 Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln
165 170 175 Ala Gln Asn
Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180
185 190 Asp Ala Gly Asp Arg Glu Thr Val
His Pro Leu Val Val Thr His Pro 195 200
205 Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr
Cys Gln Arg 210 215 220
Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225
230 235 240 Tyr Glu His Ala
Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245
250 255 Lys Asp Gln Val Leu Val Trp Asp Asn
Leu Cys Thr Met His Arg Ala 260 265
270 Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg Thr
Thr Val 275 280 285
Gly Gly Val Arg Pro Ala Arg 290 295
41888DNAArtificial SequenceSynthetic DNA sequence encoding the AAD1
protein using codons optimized for maize and with sequences
identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(888) 41atg cac gct gca ctg tca cca ctc tca cag cgc ttt
gag aga att gcg 48Met His Ala Ala Leu Ser Pro Leu Ser Gln Arg Phe
Glu Arg Ile Ala 1 5 10
15 gtc cag ccg ctg act ggc gtc ttg ggc gct gag atc acc ggc
gtc gat 96Val Gln Pro Leu Thr Gly Val Leu Gly Ala Glu Ile Thr Gly
Val Asp 20 25 30
ctg agg gag cct ctc gac gat tca acg tgg aac gaa att ctc gac gcg
144Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu Asp Ala
35 40 45 ttc
cat act tac caa gtc atc tac ttt ccc ggg caa gct att acc aac 192Phe
His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50
55 60 gaa caa cac
atc gct ttc tct cgg cga ttc ggc ccc gtc gat cca gtg 240Glu Gln His
Ile Ala Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65
70 75 80 ccc tta ctc aag tct
atc gaa ggc tac cca gag gtg cag atg ata aga 288Pro Leu Leu Lys Ser
Ile Glu Gly Tyr Pro Glu Val Gln Met Ile Arg 85
90 95 agg gag gcc aac gaa agc ggg
cgt gtg ata ggt gat gac tgg cac act 336Arg Glu Ala Asn Glu Ser Gly
Arg Val Ile Gly Asp Asp Trp His Thr 100
105 110 gac agc aca ttc ctg gat gca ccg ccg
gcc gct gtg gtg atg agg gca 384Asp Ser Thr Phe Leu Asp Ala Pro Pro
Ala Ala Val Val Met Arg Ala 115 120
125 atc gac gtg ccc gag cac gga ggt gac act ggt
ttc ttg agt atg tac 432Ile Asp Val Pro Glu His Gly Gly Asp Thr Gly
Phe Leu Ser Met Tyr 130 135 140
act gct tgg gag acg ctt tcg cct act atg caa gcc aca
atc gag ggg 480Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln Ala Thr
Ile Glu Gly 145 150 155
160 ttg aat gta gtt cac agc gcc acg cgt gtg ttc gga tct ctc tat
caa 528Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr
Gln 165 170 175
gcc caa aac cgg cgc ttt tca aat acc tcc gtc aag gtg atg gac gtt
576Ala Gln Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val
180 185 190 gac
gcg ggc gac cgt gaa acc gtg cac cct ctt gtt gta acc cat ccg 624Asp
Ala Gly Asp Arg Glu Thr Val His Pro Leu Val Val Thr His Pro
195 200 205 ggc agt ggt
cgc aag ggc cta tac gtt aac caa gtc tat tgc cag cgc 672Gly Ser Gly
Arg Lys Gly Leu Tyr Val Asn Gln Val Tyr Cys Gln Arg 210
215 220 atc gag gga atg aca
gac gca gag agt aag ccg ctc ctg caa ttc ctg 720Ile Glu Gly Met Thr
Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225 230
235 240 tac gag cac gcg aca cgg ttc
gat ttc acc tgc cgc gtg cgc tgg aaa 768Tyr Glu His Ala Thr Arg Phe
Asp Phe Thr Cys Arg Val Arg Trp Lys 245
250 255 aag gat caa gtc ctt gta tgg gac aac
ctt tgt acg atg cac cgg gcc 816Lys Asp Gln Val Leu Val Trp Asp Asn
Leu Cys Thr Met His Arg Ala 260 265
270 gtt cct gac tac gcg ggc aag ttc aga tac ctg
acg agg acc acg gtc 864Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu
Thr Arg Thr Thr Val 275 280
285 ggt gga gtt agg cca gcg aga tga
888Gly Gly Val Arg Pro Ala Arg
290 295
42295PRTArtificial SequenceSynthetic Construct 42Met His Ala Ala
Leu Ser Pro Leu Ser Gln Arg Phe Glu Arg Ile Ala 1 5
10 15 Val Gln Pro Leu Thr Gly Val Leu Gly
Ala Glu Ile Thr Gly Val Asp 20 25
30 Leu Arg Glu Pro Leu Asp Asp Ser Thr Trp Asn Glu Ile Leu
Asp Ala 35 40 45
Phe His Thr Tyr Gln Val Ile Tyr Phe Pro Gly Gln Ala Ile Thr Asn 50
55 60 Glu Gln His Ile Ala
Phe Ser Arg Arg Phe Gly Pro Val Asp Pro Val 65 70
75 80 Pro Leu Leu Lys Ser Ile Glu Gly Tyr Pro
Glu Val Gln Met Ile Arg 85 90
95 Arg Glu Ala Asn Glu Ser Gly Arg Val Ile Gly Asp Asp Trp His
Thr 100 105 110 Asp
Ser Thr Phe Leu Asp Ala Pro Pro Ala Ala Val Val Met Arg Ala 115
120 125 Ile Asp Val Pro Glu His
Gly Gly Asp Thr Gly Phe Leu Ser Met Tyr 130 135
140 Thr Ala Trp Glu Thr Leu Ser Pro Thr Met Gln
Ala Thr Ile Glu Gly 145 150 155
160 Leu Asn Val Val His Ser Ala Thr Arg Val Phe Gly Ser Leu Tyr Gln
165 170 175 Ala Gln
Asn Arg Arg Phe Ser Asn Thr Ser Val Lys Val Met Asp Val 180
185 190 Asp Ala Gly Asp Arg Glu Thr
Val His Pro Leu Val Val Thr His Pro 195 200
205 Gly Ser Gly Arg Lys Gly Leu Tyr Val Asn Gln Val
Tyr Cys Gln Arg 210 215 220
Ile Glu Gly Met Thr Asp Ala Glu Ser Lys Pro Leu Leu Gln Phe Leu 225
230 235 240 Tyr Glu His
Ala Thr Arg Phe Asp Phe Thr Cys Arg Val Arg Trp Lys 245
250 255 Lys Asp Gln Val Leu Val Trp Asp
Asn Leu Cys Thr Met His Arg Ala 260 265
270 Val Pro Asp Tyr Ala Gly Lys Phe Arg Tyr Leu Thr Arg
Thr Thr Val 275 280 285
Gly Gly Val Arg Pro Ala Arg 290 295
431368DNAAspergillus nidulansCDS(1)..(1368)Native DNA sequence encoding
Aspergillus nidulans delta-9 fatty acid desaturase protein 43atg tct
gca cca acg gcg gac atc agg gct cgc gcc ccg gag gcc aaa 48Met Ser
Ala Pro Thr Ala Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1
5 10 15 aag gtt cac atc
gct gac act gct atc aac cgc cat aac tgg tac aag 96Lys Val His Ile
Ala Asp Thr Ala Ile Asn Arg His Asn Trp Tyr Lys 20
25 30 cat gtg aac tgg ctg aac
gtt ttc ctg atc atc ggt atc ccg ctt tat 144His Val Asn Trp Leu Asn
Val Phe Leu Ile Ile Gly Ile Pro Leu Tyr 35
40 45 ggg tgc att cag gcg ttc tgg gtg
cca ctg cag ctg aag act gcc atc 192Gly Cys Ile Gln Ala Phe Trp Val
Pro Leu Gln Leu Lys Thr Ala Ile 50 55
60 tgg gcc gtc atc tac tac ttt ttc acc ggt
ctc ggt atc aca gca ggt 240Trp Ala Val Ile Tyr Tyr Phe Phe Thr Gly
Leu Gly Ile Thr Ala Gly 65 70 75
80 tac cat cgt cta tgg gct cac tgc tcg tac tcc gcc
acc ctt cct ttg 288Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser Ala
Thr Leu Pro Leu 85 90
95 cgt atc tgg ctc gct gcc gtt ggt ggt ggt gcc gtc gaa ggt
tct atc 336Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly
Ser Ile 100 105 110
cgc tgg tgg gct cgt gac cac cgc gct cac cac cgc tac acc gat acc
384Arg Trp Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr
115 120 125 gac
aaa gac ccg tac tcc gtt cgc aag ggt ctg ctc tac tct cac ctt 432Asp
Lys Asp Pro Tyr Ser Val Arg Lys Gly Leu Leu Tyr Ser His Leu 130
135 140 ggc tgg atg
gtg atg aag cag aac cct aag cgt att ggc cgt acc gat 480Gly Trp Met
Val Met Lys Gln Asn Pro Lys Arg Ile Gly Arg Thr Asp 145
150 155 160 att tcc gac ctg aac
gag gac ccc gtc gtt gtc tgg cag cac cgc aac 528Ile Ser Asp Leu Asn
Glu Asp Pro Val Val Val Trp Gln His Arg Asn 165
170 175 tac ctc aag gtc gtt ttc acg
atg gga ttg gct gtg cct atg ctt gtt 576Tyr Leu Lys Val Val Phe Thr
Met Gly Leu Ala Val Pro Met Leu Val 180
185 190 gct ggt ctt gga tgg ggt gac tgg ttg
ggc ggc ttc gtg tat gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Leu
Gly Gly Phe Val Tyr Ala Gly 195 200
205 att ctg cgt atc ttc ttc gtc cag cag gcg act
ttc tgc gtc aac tct 672Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr
Phe Cys Val Asn Ser 210 215 220
ttg gcc cac tgg ctc ggt gac cag ccc ttc gat gac cgc
aac tca cct 720Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg
Asn Ser Pro 225 230 235
240 cgt gac cac gtt atc acc gct ctc gtc acc ctt gga gag ggc tac
cac 768Arg Asp His Val Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr
His 245 250 255
aac ttc cac cac gag ttc ccc tcg gac tac cgt aac gcc atc gaa tgg
816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Glu Trp
260 265 270 cac
cag tat gat ccc acc aag tgg tcc atc tgg gcc tgg aag cag ctt 864His
Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys Gln Leu
275 280 285 ggt ctt gcc
tac gac ctg aag aag ttc cgt gcc aac gag att gag aag 912Gly Leu Ala
Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300 ggt cgt gtc cag cag
ctc cag aag aag ctt gac cgt aag cgt gcc act 960Gly Arg Val Gln Gln
Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310
315 320 ctc gat tgg ggt act cct ctt
gac cag ctc ccc gtc atg gag tgg gac 1008Leu Asp Trp Gly Thr Pro Leu
Asp Gln Leu Pro Val Met Glu Trp Asp 325
330 335 gac tac gtc gag cag gct aag aac ggc cgc
ggt ctc gtg gct att gcc 1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg
Gly Leu Val Ala Ile Ala 340 345
350 ggt gtt gtc cac gat gtc acg gac ttc atc aaa gac cac
ccc ggt ggc 1104Gly Val Val His Asp Val Thr Asp Phe Ile Lys Asp His
Pro Gly Gly 355 360 365
aag gcc atg atc agc tcc ggt att ggg aag gac gcc acc gcc atg ttc
1152Lys Ala Met Ile Ser Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe
370 375 380 aac
ggt ggt gtc tac tac cac tcc aac gcc gca cac aac ctc ctc tct 1200Asn
Gly Gly Val Tyr Tyr His Ser Asn Ala Ala His Asn Leu Leu Ser 385
390 395 400 acc atg cgt gtt
ggt gtt atc cgc ggc ggc tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val
Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp 405
410 415 aag cgt gcc cag aag gag aac
gtg gag tac gtg cgt gat ggc tct ggc 1296Lys Arg Ala Gln Lys Glu Asn
Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430 cag cgc gtc atc cgt gcc ggc gag cag
cca acc aag atc cca gaa ccc 1344Gln Arg Val Ile Arg Ala Gly Glu Gln
Pro Thr Lys Ile Pro Glu Pro 435 440
445 att ccc aca gcg gat gcg gcg tga
1368Ile Pro Thr Ala Asp Ala Ala
450 455
44455PRTAspergillus nidulans 44Met Ser Ala Pro Thr Ala Asp
Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5
10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg
His Asn Trp Tyr Lys 20 25
30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu
Tyr 35 40 45 Gly
Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50
55 60 Trp Ala Val Ile Tyr Tyr
Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70
75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser
Ala Thr Leu Pro Leu 85 90
95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile
100 105 110 Arg Trp
Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115
120 125 Asp Lys Asp Pro Tyr Ser Val
Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135
140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile
Gly Arg Thr Asp 145 150 155
160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn
165 170 175 Tyr Leu Lys
Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180
185 190 Ala Gly Leu Gly Trp Gly Asp Trp
Leu Gly Gly Phe Val Tyr Ala Gly 195 200
205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225
230 235 240 Arg Asp His Val
Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255 Asn Phe His His Glu Phe Pro Ser Asp
Tyr Arg Asn Ala Ile Glu Trp 260 265
270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys
Gln Leu 275 280 285
Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300 Gly Arg Val Gln Gln
Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310
315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu
Pro Val Met Glu Trp Asp 325 330
335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile
Ala 340 345 350 Gly
Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355
360 365 Lys Ala Met Ile Ser Ser
Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375
380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala
His Asn Leu Leu Ser 385 390 395
400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp
405 410 415 Lys Arg
Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430 Gln Arg Val Ile Arg Ala Gly
Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440
445 Ile Pro Thr Ala Asp Ala Ala 450
455 451368DNAArtificial SequenceSynthetic DNA sequence encoding
Aspergillus nidulans delta-9 fatty acid desaturase protein using
codons optimized for maize and Table 1 & Table 2 sequences are
maintainedCDS(1)..(1368) 45atg agt gca cca acg gcg gac ata agg gcg cgc
gcc ccg gag gca aaa 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg
Ala Pro Glu Ala Lys 1 5 10
15 aag gtt cac att gct gac act gct atc aat cgc cat aac
tgg tat aag 96Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn
Trp Tyr Lys 20 25 30
cat gtg aat tgg ctg aac gtt ttt ctg atc atc ggc atc ccg ctt
tat 144His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu
Tyr 35 40 45
ggg tgt att caa gcg ttc tgg gtg cca ctc cag ctc aag act gcc atc
192Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile
50 55 60 tgg
gcc gta atc tac tac ttc ttt acc ggt ttg gga atc aca gcg ggt 240Trp
Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65
70 75 80 tat cac aga
ttg tgg gca cac tgc tcg tac tcc gcc acc ctt cct tta 288Tyr His Arg
Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu
85 90 95 cgt ata tgg ctc gct
gcc gta gga gga ggc gcc gtc gaa ggt tca atc 336Arg Ile Trp Leu Ala
Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100
105 110 cgt tgg tgg gct aga gac cat
cgt gct cat cat aga tat acc gat aca 384Arg Trp Trp Ala Arg Asp His
Arg Ala His His Arg Tyr Thr Asp Thr 115 120
125 gac aaa gac ccg tac tcc gtt cgc aag
ggg ctg cta tac tct cac ctt 432Asp Lys Asp Pro Tyr Ser Val Arg Lys
Gly Leu Leu Tyr Ser His Leu 130 135
140 ggc tgg atg gtg atg aag cag aac cct aag cgt
att ggc aga acc gat 480Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg
Ile Gly Arg Thr Asp 145 150 155
160 att tcc gac ctg aac gag gac ccc gtc gtt gtc tgg cag
cac cgg aac 528Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln
His Arg Asn 165 170
175 tac ctc aag gtc gtt ttc acg atg gga ttg gct gtg cct atg ctt
gtt 576Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu
Val 180 185 190
gct ggg ctt ggc tgg gga gac tgg ttg ggc ggc ttc gtg tat gcc ggc
624Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly
195 200 205 ata
ctg aga atc ttt ttc gtc cag caa gcg act ttt tgc gtc aac tct 672Ile
Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210
215 220 ttg gcc cac
tgg ctc gga gat cag ccg ttc gat gac cgg aac agt cct 720Leu Ala His
Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225
230 235 240 agg gac cac gtt atc
act gct ctc gtc acc cta gga gag ggc tac cac 768Arg Asp His Val Ile
Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255 aac ttc cat cac gag ttc ccc
tcg gac tac cgg aac gcc atc gaa tgg 816Asn Phe His His Glu Phe Pro
Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260
265 270 cac cag tat gat cca acg aag tgg agc
atc tgg gcc tgg aag cag ctt 864His Gln Tyr Asp Pro Thr Lys Trp Ser
Ile Trp Ala Trp Lys Gln Leu 275 280
285 ggt tta gcc tac gac ctg aag aaa ttc aga gcc
aac gag att gag aaa 912Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala
Asn Glu Ile Glu Lys 290 295 300
ggg cgt gtc caa cag ctg caa aag aaa ctg gac cgt aag
cgg gcg act 960Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys
Arg Ala Thr 305 310 315
320 ctc gat tgg gga aca cct ctg gat cag ctc ccc gtc atg gag tgg
gac 1008Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp
Asp 325 330 335
gac tac gtg gag caa gca aag aac ggt cgc ggt ctc gtg gca ata gcg
1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala
340 345 350 ggc gtg
gtg cac gat gtc acg gat ttc atc aaa gat cac ccg ggg ggc 1104Gly Val
Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355
360 365 aag gcc atg atc agc
tcc ggg att ggc aag gac gca acc gcc atg ttc 1152Lys Ala Met Ile Ser
Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370
375 380 aat ggg gga gtc tac tac cac
agc aac gca gca cac aat ctc ttg tca 1200Asn Gly Gly Val Tyr Tyr His
Ser Asn Ala Ala His Asn Leu Leu Ser 385 390
395 400 aca atg agg gtg ggt gtt att agg ggc ggc
tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val Gly Val Ile Arg Gly Gly
Cys Glu Val Glu Ile Trp 405 410
415 aag agg gcg caa aag gag aat gtg gag tac gtg cga gat
ggc tct ggt 1296Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp
Gly Ser Gly 420 425 430
caa cgc gtg atc aga gcg ggc gag cag cca acc aag ata cca gaa ccg
1344Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro
435 440 445 att
ccc aca gcg gat gcg gcg tag 1368Ile
Pro Thr Ala Asp Ala Ala 450
455
46455PRTArtificial SequenceSynthetic Construct 46Met Ser Ala Pro Thr Ala
Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5
10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg
His Asn Trp Tyr Lys 20 25
30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu
Tyr 35 40 45 Gly
Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50
55 60 Trp Ala Val Ile Tyr Tyr
Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70
75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser
Ala Thr Leu Pro Leu 85 90
95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile
100 105 110 Arg Trp
Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115
120 125 Asp Lys Asp Pro Tyr Ser Val
Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135
140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile
Gly Arg Thr Asp 145 150 155
160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn
165 170 175 Tyr Leu Lys
Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180
185 190 Ala Gly Leu Gly Trp Gly Asp Trp
Leu Gly Gly Phe Val Tyr Ala Gly 195 200
205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225
230 235 240 Arg Asp His Val
Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255 Asn Phe His His Glu Phe Pro Ser Asp
Tyr Arg Asn Ala Ile Glu Trp 260 265
270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys
Gln Leu 275 280 285
Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300 Gly Arg Val Gln Gln
Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310
315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu
Pro Val Met Glu Trp Asp 325 330
335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile
Ala 340 345 350 Gly
Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355
360 365 Lys Ala Met Ile Ser Ser
Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375
380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala
His Asn Leu Leu Ser 385 390 395
400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp
405 410 415 Lys Arg
Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430 Gln Arg Val Ile Arg Ala Gly
Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440
445 Ile Pro Thr Ala Asp Ala Ala 450
455 471368DNAArtificial SequenceSynthetic DNA sequence in accordance
with the invention encoding Aspergillus nidulans delta-9 fatty acid
desaturase protein using codons optimized for maize and with
sequences identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(1368) 47atg agt gca cca acg gcg gac ata agg gcg cgc
gcc ccg gag gca aaa 48Met Ser Ala Pro Thr Ala Asp Ile Arg Ala Arg
Ala Pro Glu Ala Lys 1 5 10
15 aag gtt cac att gct gac act gct atc aat cgc cat aac
tgg tat aag 96Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg His Asn
Trp Tyr Lys 20 25 30
cat gtg aat tgg ctg aac gtt ttt ctg atc atc ggc atc ccg ctt
tat 144His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu
Tyr 35 40 45
ggg tgt att caa gcg ttc tgg gtg cca ctc cag ctc aag act gcc atc
192Gly Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile
50 55 60 tgg
gcc gta atc tac tac ttc ttt acc ggt ttg gga atc aca gcg ggt 240Trp
Ala Val Ile Tyr Tyr Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65
70 75 80 tat cac aga
ttg tgg gca cac tgc tcg tac tcc gcc acc ctt cct tta 288Tyr His Arg
Leu Trp Ala His Cys Ser Tyr Ser Ala Thr Leu Pro Leu
85 90 95 cgt ata tgg ctc gct
gcc gta gga gga ggc gcc gtc gaa ggt tca atc 336Arg Ile Trp Leu Ala
Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile 100
105 110 cgt tgg tgg gct aga gac cat
cgt gct cat cat aga tat acc gat aca 384Arg Trp Trp Ala Arg Asp His
Arg Ala His His Arg Tyr Thr Asp Thr 115 120
125 gac aaa gac ccg tac tcc gtt cgc aag
ggg ctg cta tac tct cac ctt 432Asp Lys Asp Pro Tyr Ser Val Arg Lys
Gly Leu Leu Tyr Ser His Leu 130 135
140 ggc tgg atg gtg atg aag cag aac cct aag cgt
att ggc aga acc gat 480Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg
Ile Gly Arg Thr Asp 145 150 155
160 att agc gac ctg aac gag gac ccc gtc gtt gtc tgg cag
cac cgg aac 528Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln
His Arg Asn 165 170
175 tac ctc aag gtc gtt ttc acg atg gga ttg gct gtg cct atg ctt
gtt 576Tyr Leu Lys Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu
Val 180 185 190
gct ggg ctt ggc tgg gga gac tgg ttg ggc ggc ttc gtg tat gcc ggc
624Ala Gly Leu Gly Trp Gly Asp Trp Leu Gly Gly Phe Val Tyr Ala Gly
195 200 205 ata
ctg aga atc ttt ttc gtc cag caa gcg act ttt tgc gtc aac tct 672Ile
Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys Val Asn Ser 210
215 220 ttg gcc cac
tgg ctc gga gat cag ccg ttc gat gac cgg aac agt cct 720Leu Ala His
Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225
230 235 240 agg gac cac gtt atc
act gct ctc gtc acc cta gga gag ggc tac cac 768Arg Asp His Val Ile
Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255 aac ttc cat cac gag ttc ccc
tcg gac tac cgg aac gcc atc gaa tgg 816Asn Phe His His Glu Phe Pro
Ser Asp Tyr Arg Asn Ala Ile Glu Trp 260
265 270 cac cag tat gat cca acg aag tgg agc
atc tgg gcc tgg aag cag ctt 864His Gln Tyr Asp Pro Thr Lys Trp Ser
Ile Trp Ala Trp Lys Gln Leu 275 280
285 ggt tta gcc tac gac ctg aag aaa ttc aga gcc
aac gag att gag aaa 912Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala
Asn Glu Ile Glu Lys 290 295 300
ggg cgt gtc caa cag ctg caa aag aaa ctg gac cgt aag
cgg gcg act 960Gly Arg Val Gln Gln Leu Gln Lys Lys Leu Asp Arg Lys
Arg Ala Thr 305 310 315
320 ctc gat tgg gga aca cct ctg gat cag ctc ccc gtc atg gag tgg
gac 1008Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu Pro Val Met Glu Trp
Asp 325 330 335
gac tac gtg gag caa gca aag aac ggt cgc ggt ctc gtg gca ata gcg
1056Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile Ala
340 345 350 ggc gtg
gtg cac gat gtc acg gat ttc atc aaa gat cac ccg ggg ggc 1104Gly Val
Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355
360 365 aag gcc atg atc agc
tcc ggg att ggc aag gac gca acc gcc atg ttc 1152Lys Ala Met Ile Ser
Ser Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370
375 380 aat ggg gga gtc tac tac cac
agc aac gca gca cac aat ctc ttg tca 1200Asn Gly Gly Val Tyr Tyr His
Ser Asn Ala Ala His Asn Leu Leu Ser 385 390
395 400 aca atg agg gtg ggt gtt att agg ggc ggc
tgt gaa gtc gaa atc tgg 1248Thr Met Arg Val Gly Val Ile Arg Gly Gly
Cys Glu Val Glu Ile Trp 405 410
415 aag agg gcg caa aag gag aat gtg gag tac gtg cga gat
ggc tct ggt 1296Lys Arg Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp
Gly Ser Gly 420 425 430
caa cgc gtg atc aga gcg ggc gag cag cca acc aag ata cca gaa ccg
1344Gln Arg Val Ile Arg Ala Gly Glu Gln Pro Thr Lys Ile Pro Glu Pro
435 440 445 att
ccc aca gcg gat gcg gcg tag 1368Ile
Pro Thr Ala Asp Ala Ala 450
455
48455PRTArtificial SequenceSynthetic Construct 48Met Ser Ala Pro Thr Ala
Asp Ile Arg Ala Arg Ala Pro Glu Ala Lys 1 5
10 15 Lys Val His Ile Ala Asp Thr Ala Ile Asn Arg
His Asn Trp Tyr Lys 20 25
30 His Val Asn Trp Leu Asn Val Phe Leu Ile Ile Gly Ile Pro Leu
Tyr 35 40 45 Gly
Cys Ile Gln Ala Phe Trp Val Pro Leu Gln Leu Lys Thr Ala Ile 50
55 60 Trp Ala Val Ile Tyr Tyr
Phe Phe Thr Gly Leu Gly Ile Thr Ala Gly 65 70
75 80 Tyr His Arg Leu Trp Ala His Cys Ser Tyr Ser
Ala Thr Leu Pro Leu 85 90
95 Arg Ile Trp Leu Ala Ala Val Gly Gly Gly Ala Val Glu Gly Ser Ile
100 105 110 Arg Trp
Trp Ala Arg Asp His Arg Ala His His Arg Tyr Thr Asp Thr 115
120 125 Asp Lys Asp Pro Tyr Ser Val
Arg Lys Gly Leu Leu Tyr Ser His Leu 130 135
140 Gly Trp Met Val Met Lys Gln Asn Pro Lys Arg Ile
Gly Arg Thr Asp 145 150 155
160 Ile Ser Asp Leu Asn Glu Asp Pro Val Val Val Trp Gln His Arg Asn
165 170 175 Tyr Leu Lys
Val Val Phe Thr Met Gly Leu Ala Val Pro Met Leu Val 180
185 190 Ala Gly Leu Gly Trp Gly Asp Trp
Leu Gly Gly Phe Val Tyr Ala Gly 195 200
205 Ile Leu Arg Ile Phe Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
Leu Ala His Trp Leu Gly Asp Gln Pro Phe Asp Asp Arg Asn Ser Pro 225
230 235 240 Arg Asp His Val
Ile Thr Ala Leu Val Thr Leu Gly Glu Gly Tyr His 245
250 255 Asn Phe His His Glu Phe Pro Ser Asp
Tyr Arg Asn Ala Ile Glu Trp 260 265
270 His Gln Tyr Asp Pro Thr Lys Trp Ser Ile Trp Ala Trp Lys
Gln Leu 275 280 285
Gly Leu Ala Tyr Asp Leu Lys Lys Phe Arg Ala Asn Glu Ile Glu Lys 290
295 300 Gly Arg Val Gln Gln
Leu Gln Lys Lys Leu Asp Arg Lys Arg Ala Thr 305 310
315 320 Leu Asp Trp Gly Thr Pro Leu Asp Gln Leu
Pro Val Met Glu Trp Asp 325 330
335 Asp Tyr Val Glu Gln Ala Lys Asn Gly Arg Gly Leu Val Ala Ile
Ala 340 345 350 Gly
Val Val His Asp Val Thr Asp Phe Ile Lys Asp His Pro Gly Gly 355
360 365 Lys Ala Met Ile Ser Ser
Gly Ile Gly Lys Asp Ala Thr Ala Met Phe 370 375
380 Asn Gly Gly Val Tyr Tyr His Ser Asn Ala Ala
His Asn Leu Leu Ser 385 390 395
400 Thr Met Arg Val Gly Val Ile Arg Gly Gly Cys Glu Val Glu Ile Trp
405 410 415 Lys Arg
Ala Gln Lys Glu Asn Val Glu Tyr Val Arg Asp Gly Ser Gly 420
425 430 Gln Arg Val Ile Arg Ala Gly
Glu Gln Pro Thr Lys Ile Pro Glu Pro 435 440
445 Ile Pro Thr Ala Asp Ala Ala 450
455 49798DNAXerophyta viscosaCDS(1)..(798)Native DNA sequence
encoding Xerophyta viscosa SAP1 protein 49atg agg aac gag ggt ttt
ctg aaa atg aag acc gac gtt gga gtc gcc 48Met Arg Asn Glu Gly Phe
Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5
10 15 gac gag gtg atc tcc gga gat ctc
aag cag ctt ggt gac gct gca aag 96Asp Glu Val Ile Ser Gly Asp Leu
Lys Gln Leu Gly Asp Ala Ala Lys 20 25
30 cgg cta gct aaa cat gcg atc aag ctc ggc
gcc agc ttc ggg gtt ggc 144Arg Leu Ala Lys His Ala Ile Lys Leu Gly
Ala Ser Phe Gly Val Gly 35 40
45 tct acc ata gtc cag gct att gct tcg atc gct gct
atc tat ttg ttg 192Ser Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala
Ile Tyr Leu Leu 50 55 60
ata ttg gac cgg aca aac tgg cgt aca aat atc ttg aca tca
ctt cta 240Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser
Leu Leu 65 70 75
80 att cca tat gtt tac ttg agt ctt cct tca gtg ata ttc aac cta ttc
288Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe
85 90 95
agg ggc gac ctg ggc aga tgg ctt tca ttc att ggc gta gta atg aag
336Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys
100 105 110 ctc
ttc ttc cac cga cac ttc cca gtt acc ttg gaa ctg ctt gtg tct 384Leu
Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser
115 120 125 ctc att ctc
ctg att gtg gtt tcc ccc act ttc att gcc cac aca atc 432Leu Ile Leu
Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130
135 140 aga ggc agt ctc att
gga gtc ttc atc ttc ctt gtc atc gcc tgc tac 480Arg Gly Ser Leu Ile
Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150
155 160 ctc ctc caa gag cac att aga
tca gct ggt ggc ttc aaa aac gcg ttc 528Leu Leu Gln Glu His Ile Arg
Ser Ala Gly Gly Phe Lys Asn Ala Phe 165
170 175 aca aag agc aat ggg att tca aac agc
gtc ggg atc atc att cta ctg 576Thr Lys Ser Asn Gly Ile Ser Asn Ser
Val Gly Ile Ile Ile Leu Leu 180 185
190 atc cac ccg atc tgg agc ttg gtg gtg tat ttc
ctc tac acg tct ttg 624Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe
Leu Tyr Thr Ser Leu 195 200
205 ctg caa ctt ctt gca tac tct cct tcc cct tgt tgt tgc
ata tta tac 672Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys
Ile Leu Tyr 210 215 220
aat aag tgg ttt aat ttc atg cat gtt tgt aaa tgt gta agc ctt
cat 720Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu
His 225 230 235 240
atg tat tct cag tca att ggg tca tgc gtg tcc ata ttt ttc gtg cag
768Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln
245 250 255 ttt
gta ttc atc tat gaa gct gaa ttt taa 798Phe
Val Phe Ile Tyr Glu Ala Glu Phe
260 265
50265PRTXerophyta viscosa 50Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr
Asp Val Gly Val Ala 1 5 10
15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys
20 25 30 Arg Leu
Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly 35
40 45 Ser Thr Ile Val Gln Ala Ile
Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50 55
60 Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu
Thr Ser Leu Leu 65 70 75
80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe
85 90 95 Arg Gly Asp
Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys 100
105 110 Leu Phe Phe His Arg His Phe Pro
Val Thr Leu Glu Leu Leu Val Ser 115 120
125 Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe Ile Ala
His Thr Ile 130 135 140
Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145
150 155 160 Leu Leu Gln Glu
His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe 165
170 175 Thr Lys Ser Asn Gly Ile Ser Asn Ser
Val Gly Ile Ile Ile Leu Leu 180 185
190 Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr
Ser Leu 195 200 205
Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210
215 220 Asn Lys Trp Phe Asn
Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230
235 240 Met Tyr Ser Gln Ser Ile Gly Ser Cys Val
Ser Ile Phe Phe Val Gln 245 250
255 Phe Val Phe Ile Tyr Glu Ala Glu Phe 260
265 51798DNAArtificial SequenceSynthetic DNA sequence encoding
Xerophyta viscosa SAP1 protein using codons optimized for maize and
Table 1 & Table 2 sequences are maintainedCDS(1)..(798) 51atg aga aac gaa
ggt ttt ctg aag atg aaa acg gac gtt ggg gtt gct 48Met Arg Asn Glu
Gly Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5
10 15 gac gaa gtc atc agc ggt
gat ttg aag cag ttg ggt gat gct gcc aaa 96Asp Glu Val Ile Ser Gly
Asp Leu Lys Gln Leu Gly Asp Ala Ala Lys 20
25 30 cgc ctt gct aag cac gct atc aaa
ctg gga gcc agc ttt ggt gtt ggt 144Arg Leu Ala Lys His Ala Ile Lys
Leu Gly Ala Ser Phe Gly Val Gly 35 40
45 tca act atc gtt caa gcc atc gca tca ata
gca gcc atc tat ctt ctg 192Ser Thr Ile Val Gln Ala Ile Ala Ser Ile
Ala Ala Ile Tyr Leu Leu 50 55
60 att ctc gat agg acc aac tgg agg acc aac atc ttg
acg tcc ctc ctc 240Ile Leu Asp Arg Thr Asn Trp Arg Thr Asn Ile Leu
Thr Ser Leu Leu 65 70 75
80 att ccc tac gtg tat ctg tcc ctc ccg agc gtc atc ttc aat
ctc ttt 288Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val Ile Phe Asn
Leu Phe 85 90 95
cgt ggg gac ctc ggg aga tgg ctg tca ttc ata ggc gtt gtg atg aag
336Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys
100 105 110 ctg
ttc ttt cat agg cac ttt cct gtt act ttg gag ctg ctt gtg agc 384Leu
Phe Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser
115 120 125 ctc att ctt
ttg att gtc gtg tca cct acc ttc ata gct cat aca att 432Leu Ile Leu
Leu Ile Val Val Ser Pro Thr Phe Ile Ala His Thr Ile 130
135 140 cgt gga tct ttg att
ggg gtg ttc atc ttc ttg gtg ata gca tgt tat 480Arg Gly Ser Leu Ile
Gly Val Phe Ile Phe Leu Val Ile Ala Cys Tyr 145 150
155 160 ctg ctt caa gag cac att aga
tca gct ggt ggc ttc aag aac gcc ttt 528Leu Leu Gln Glu His Ile Arg
Ser Ala Gly Gly Phe Lys Asn Ala Phe 165
170 175 aca aag tct aat gga atc tcc aac agc
gtg ggc atc atc atc ctt ctg 576Thr Lys Ser Asn Gly Ile Ser Asn Ser
Val Gly Ile Ile Ile Leu Leu 180 185
190 atc cac ccg att tgg tct ctc gtc gtc tac ttc
ctc tac act tca ctt 624Ile His Pro Ile Trp Ser Leu Val Val Tyr Phe
Leu Tyr Thr Ser Leu 195 200
205 ctc cag ctt ttg gcc tac tca cca tcc ccg tgc tgc tgc
ata tta tac 672Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys
Ile Leu Tyr 210 215 220
aac aag tgg ttc aac ttc atg cat gtt tgc aag tgc gtc tct ttg
cac 720Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu
His 225 230 235 240
atg tac tct cag tcc ata ggc tca tgt gtt tca ata ttt ttc gtc cag
768Met Tyr Ser Gln Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln
245 250 255 ttc
gtg ttc atc tat gag gct gag ttt taa 798Phe
Val Phe Ile Tyr Glu Ala Glu Phe
260 265
52265PRTArtificial SequenceSynthetic Construct 52Met Arg Asn Glu Gly Phe
Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5
10 15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu
Gly Asp Ala Ala Lys 20 25
30 Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val
Gly 35 40 45 Ser
Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50
55 60 Ile Leu Asp Arg Thr Asn
Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70
75 80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val
Ile Phe Asn Leu Phe 85 90
95 Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys
100 105 110 Leu Phe
Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115
120 125 Leu Ile Leu Leu Ile Val Val
Ser Pro Thr Phe Ile Ala His Thr Ile 130 135
140 Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val
Ile Ala Cys Tyr 145 150 155
160 Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe
165 170 175 Thr Lys Ser
Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180
185 190 Ile His Pro Ile Trp Ser Leu Val
Val Tyr Phe Leu Tyr Thr Ser Leu 195 200
205 Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys
Ile Leu Tyr 210 215 220
Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225
230 235 240 Met Tyr Ser Gln
Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245
250 255 Phe Val Phe Ile Tyr Glu Ala Glu Phe
260 265 53798DNAArtificial SequenceSynthetic
DNA sequence in accordance with the invention encoding Xerophyta
viscosa SAP1 protein using codons optimized for maize and with
sequences identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(798) 53atg aga aac gaa ggt ttt ctg aag atg aaa acg gac
gtt ggg gtt gct 48Met Arg Asn Glu Gly Phe Leu Lys Met Lys Thr Asp
Val Gly Val Ala 1 5 10
15 gac gaa gtc atc agc ggt gat ttg aag cag ttg ggt gat gct
gcc aaa 96Asp Glu Val Ile Ser Gly Asp Leu Lys Gln Leu Gly Asp Ala
Ala Lys 20 25 30
cgc ctt gct aag cac gct atc aaa ctg gga gcc agc ttt ggt gtt ggt
144Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val Gly
35 40 45 tca
act atc gtt caa gcc atc gca tca ata gca gcc atc tat ctt ctg 192Ser
Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50
55 60 att ctc gat
agg acc aac tgg agg acc aac atc ttg acg tcc ctc ctc 240Ile Leu Asp
Arg Thr Asn Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65
70 75 80 att ccc tac gtg tat
ctg tcc ctc ccg agc gtc atc ttc aat ctc ttt 288Ile Pro Tyr Val Tyr
Leu Ser Leu Pro Ser Val Ile Phe Asn Leu Phe 85
90 95 cgt ggg gac ctc ggg aga tgg
ctg tca ttc ata ggc gtt gtg atg aag 336Arg Gly Asp Leu Gly Arg Trp
Leu Ser Phe Ile Gly Val Val Met Lys 100
105 110 ctg ttc ttt cat agg cac ttt cct gtt
act ttg gag ctg ctt gtg agc 384Leu Phe Phe His Arg His Phe Pro Val
Thr Leu Glu Leu Leu Val Ser 115 120
125 ctc att ctt ttg att gtc gtg tct cct acc ttc
ata gct cat aca att 432Leu Ile Leu Leu Ile Val Val Ser Pro Thr Phe
Ile Ala His Thr Ile 130 135 140
cgt gga tct ttg att ggg gtg ttc atc ttc ttg gtg ata
gca tgt tat 480Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val Ile
Ala Cys Tyr 145 150 155
160 ctg ctt caa gag cac att aga tca gct ggt ggc ttc aag aac gcc
ttt 528Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala
Phe 165 170 175
aca aag tct aat gga atc tcc aac agc gtg ggc atc atc atc ctt ctg
576Thr Lys Ser Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu
180 185 190 atc
cac ccg att tgg tct ctc gtc gtc tac ttc ctc tac act tca ctt 624Ile
His Pro Ile Trp Ser Leu Val Val Tyr Phe Leu Tyr Thr Ser Leu
195 200 205 ctc cag ctt
ttg gcc tac tca cca tcc cca tgc tgc tgt att ctt tac 672Leu Gln Leu
Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys Ile Leu Tyr 210
215 220 aac aaa tgg ttc aac
ttc atg cac gtg tgc aag tgc gtc tct ttg cac 720Asn Lys Trp Phe Asn
Phe Met His Val Cys Lys Cys Val Ser Leu His 225 230
235 240 atg tac tct cag tcc att ggc
tca tgt gtt tca atc ttc ttt gtc cag 768Met Tyr Ser Gln Ser Ile Gly
Ser Cys Val Ser Ile Phe Phe Val Gln 245
250 255 ttc gtg ttc atc tat gag gct gag ttt
taa 798Phe Val Phe Ile Tyr Glu Ala Glu Phe
260
26554265PRTArtificial SequenceSynthetic Construct 54Met Arg Asn Glu Gly
Phe Leu Lys Met Lys Thr Asp Val Gly Val Ala 1 5
10 15 Asp Glu Val Ile Ser Gly Asp Leu Lys Gln
Leu Gly Asp Ala Ala Lys 20 25
30 Arg Leu Ala Lys His Ala Ile Lys Leu Gly Ala Ser Phe Gly Val
Gly 35 40 45 Ser
Thr Ile Val Gln Ala Ile Ala Ser Ile Ala Ala Ile Tyr Leu Leu 50
55 60 Ile Leu Asp Arg Thr Asn
Trp Arg Thr Asn Ile Leu Thr Ser Leu Leu 65 70
75 80 Ile Pro Tyr Val Tyr Leu Ser Leu Pro Ser Val
Ile Phe Asn Leu Phe 85 90
95 Arg Gly Asp Leu Gly Arg Trp Leu Ser Phe Ile Gly Val Val Met Lys
100 105 110 Leu Phe
Phe His Arg His Phe Pro Val Thr Leu Glu Leu Leu Val Ser 115
120 125 Leu Ile Leu Leu Ile Val Val
Ser Pro Thr Phe Ile Ala His Thr Ile 130 135
140 Arg Gly Ser Leu Ile Gly Val Phe Ile Phe Leu Val
Ile Ala Cys Tyr 145 150 155
160 Leu Leu Gln Glu His Ile Arg Ser Ala Gly Gly Phe Lys Asn Ala Phe
165 170 175 Thr Lys Ser
Asn Gly Ile Ser Asn Ser Val Gly Ile Ile Ile Leu Leu 180
185 190 Ile His Pro Ile Trp Ser Leu Val
Val Tyr Phe Leu Tyr Thr Ser Leu 195 200
205 Leu Gln Leu Leu Ala Tyr Ser Pro Ser Pro Cys Cys Cys
Ile Leu Tyr 210 215 220
Asn Lys Trp Phe Asn Phe Met His Val Cys Lys Cys Val Ser Leu His 225
230 235 240 Met Tyr Ser Gln
Ser Ile Gly Ser Cys Val Ser Ile Phe Phe Val Gln 245
250 255 Phe Val Phe Ile Tyr Glu Ala Glu Phe
260 265 55717DNAAequorea
victoriaCDS(1)..(717)Native DNA sequence encoding Aequorea victoria
GFP1 protein 55atg agt aaa gga gaa gaa ctt ttc act gga gtg gtc cca gtt
ctt gtt 48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Val
Leu Val 1 5 10 15
gaa tta gat ggc gat gtt aat ggg caa aaa ttc tct gtc agt gga gag
96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu
20 25 30 ggt
gaa ggt gat gca aca tac gga aaa ctt acc ctt aat ttt att tgc 144Gly
Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys
35 40 45 act act ggg
aag cta cct gtt cca tgg cca aca ctt gtc act act ttc 192Thr Thr Gly
Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 tct tat ggt gtt caa
tgc ttc tca aga tac cca gat cat atg aaa cag 240Ser Tyr Gly Val Gln
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70
75 80 cat gac ttt ttc aag agt gcc
atg ccc gaa ggt tat gta cag gaa aga 288His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu Arg 85
90 95 act ata ttt tac aaa gat gac ggg aac
tac aag aca cgt gct gaa gtc 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn
Tyr Lys Thr Arg Ala Glu Val 100 105
110 aag ttt gaa ggt gat acc ctt gtt aat aga atc
gag tta aaa ggt att 384Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile
Glu Leu Lys Gly Ile 115 120
125 gat ttt aaa gaa gat gga aac att ctt gga cac aaa atg
gaa tac aac 432Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met
Glu Tyr Asn 130 135 140
tat aac tca cat aat gta tac atc atg gga gac aaa cca aag aat
ggc 480Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn
Gly 145 150 155 160
atc aaa gtt aac ttc aaa att aga cac aac att aaa gat gga agc gtt
528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175 caa
tta gca gac cat tat caa caa aat act cca att ggc gat ggc cct 576Gln
Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190 gtc ctt tta
cca gac aac cat tac ctg tcc aca caa tct gcc ctt tcc 624Val Leu Leu
Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195
200 205 aaa gat ccc aac gaa
aag aga gat cac atg atc ctt ctt gag ttt gta 672Lys Asp Pro Asn Glu
Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210
215 220 aca gct gct agg att aca cat
ggc atg gat gaa cta tac aaa taa 717Thr Ala Ala Arg Ile Thr His
Gly Met Asp Glu Leu Tyr Lys 225 230
235 56238PRTAequorea victoria 56Met Ser Lys Gly Glu Glu Leu
Phe Thr Gly Val Val Pro Val Leu Val 1 5
10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe
Ser Val Ser Gly Glu 20 25
30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile
Cys 35 40 45 Thr
Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 Ser Tyr Gly Val Gln Cys
Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70
75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly
Tyr Val Gln Glu Arg 85 90
95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
100 105 110 Lys Phe
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125 Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Met Glu Tyr Asn 130 135
140 Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys
Pro Lys Asn Gly 145 150 155
160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175 Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190 Val Leu Leu Pro Asp Asn His Tyr
Leu Ser Thr Gln Ser Ala Leu Ser 195 200
205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu
Glu Phe Val 210 215 220
Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225
230 235 57717DNAArtificial
SequenceSynthetic DNA sequence encoding Aequorea victoria GFP1
protein using codons optimized for maize and Table 1 & Table 2
sequences are maintainedCDS(1)..(717) 57atg agt aaa ggg gaa gaa ctt ttc
acc ggc gtg gtc cca gtc ctc gtt 48Met Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Val Leu Val 1 5
10 15 gag ttg gat ggc gat gtg aat ggg caa aaa
ttc tct gtc tcc ggg gag 96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys
Phe Ser Val Ser Gly Glu 20 25
30 ggt gag ggt gat gca acc tac gga aag ctg acc cta
aat ttt att tgc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu
Asn Phe Ile Cys 35 40 45
acg act ggg aag ttg cct gtg cct tgg ccg aca ctg gtg acg
acg ttc 192Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
Thr Phe 50 55 60
tct tat ggt gtg cag tgt ttc tca cgc tac ccg gat cat atg aaa cag
240Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
65 70 75 80 cat
gac ttt ttc aag tcg gcc atg cca gaa ggc tat gta caa gag aga 288His
Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
85 90 95 act ata ttt
tac aag gac gac ggg aac tac aag aca cgt gct gag gtg 336Thr Ile Phe
Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100
105 110 aag ttc gag ggt gat
acc ctt gtt aat cgg atc gag cta aag ggc att 384Lys Phe Glu Gly Asp
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125 gac ttt aag gag gac gga aac
att ctg gga cac aaa atg gaa tac aac 432Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Met Glu Tyr Asn 130 135
140 tat aac tcg cac aac gta tac atc atg
gga gac aaa cca aag aat ggc 480Tyr Asn Ser His Asn Val Tyr Ile Met
Gly Asp Lys Pro Lys Asn Gly 145 150
155 160 ata aag gtt aac ttc aag att cga cac aac att
aaa gac ggc agc gtt 528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile
Lys Asp Gly Ser Val 165 170
175 cag ttg gcc gac cac tat caa caa aat act cca att ggc
gat ggc cct 576Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
Asp Gly Pro 180 185 190
gtc ctc tta ccc gac aac cat tac ctg tcc acg caa tca gcg ctc
agc 624Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
Ser 195 200 205
aag gac ccc aac gag aag agg gat cac atg atc ctc ctt gag ttt gtc
672Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val
210 215 220 acc
gca gct agg ata acc cac ggc atg gat gaa ctg tac aag taa 717Thr
Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225
230 235 58238PRTArtificial
SequenceSynthetic Construct 58Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val
Val Pro Val Leu Val 1 5 10
15 Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu
20 25 30 Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35
40 45 Thr Thr Gly Lys Leu Pro Val
Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55
60 Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
His Met Lys Gln 65 70 75
80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
85 90 95 Thr Ile Phe
Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100
105 110 Lys Phe Glu Gly Asp Thr Leu Val
Asn Arg Ile Glu Leu Lys Gly Ile 115 120
125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met
Glu Tyr Asn 130 135 140
Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly 145
150 155 160 Ile Lys Val Asn
Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165
170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn
Thr Pro Ile Gly Asp Gly Pro 180 185
190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205
Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu Leu Glu Phe Val 210
215 220 Thr Ala Ala Arg Ile
Thr His Gly Met Asp Glu Leu Tyr Lys 225 230
235 59717DNAArtificial SequenceSynthetic DNA sequence in
accordance with the invention encoding Aequorea victoria GFP1
protein using codons optimized for maize and with sequences
identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(717) 59atg agt aaa ggg gaa gaa ctt ttc acc ggc gtg gtc
cca gtc ctc gtt 48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val
Pro Val Leu Val1 5 10 15
gag ttg gat ggc gat gtg aat ggg caa aaa ttc tct gtc tcc ggg gag
96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Ser Gly Glu
20 25 30 ggt gag
ggt gat gca acc tac gga aag ctg acc cta aat ttc atc tgc 144Gly Glu
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe Ile Cys 35
40 45 acg act ggg aag
ttg cct gtg cct tgg ccg aca ctg gtg acg acg ttc 192Thr Thr Gly Lys
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 tct tat ggt gtg cag tgt
ttc tca cgc tac ccg gat cat atg aaa cag 240Ser Tyr Gly Val Gln Cys
Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70
75 80 cat gac ttt ttc aag tcg gcc atg
cca gaa ggc tat gta caa gag aga 288His Asp Phe Phe Lys Ser Ala Met
Pro Glu Gly Tyr Val Gln Glu Arg 85
90 95 act atc ttt tac aag gac gac ggg aac tac
aag aca cgt gct gag gtg 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr
Lys Thr Arg Ala Glu Val 100 105
110 aag ttc gag ggt gat acc ctt gtt aat cgg atc gag
cta aag ggc att 384Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu
Leu Lys Gly Ile 115 120 125
gac ttt aag gag gac gga aac att ctg gga cac aaa atg gaa
tac aac 432Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Met Glu
Tyr Asn 130 135 140
tat aac tcg cac aac gta tac atc atg gga gac aaa cca aag aat ggc
480Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly
145 150 155 160 ata
aag gtt aac ttc aag att cga cac aac att aaa gac ggc agc gtt 528Ile
Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175 cag ttg gcc
gac cac tat caa caa aat act cca att ggc gat ggc cct 576Gln Leu Ala
Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190 gtc ctc tta ccc gac
aac cat tac ctg tcc acg caa tca gcg ctc agc 624Val Leu Leu Pro Asp
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195
200 205 aag gac ccc aac gag aag agg
gat cac atg atc ctc ctt gag ttt gtc 672Lys Asp Pro Asn Glu Lys Arg
Asp His Met Ile Leu Leu Glu Phe Val 210 215
220 acc gca gct agg ata acc cac ggc atg
gat gaa ctg tac aag taa 717Thr Ala Ala Arg Ile Thr His Gly Met
Asp Glu Leu Tyr Lys 225 230 235
60238PRTArtificial SequenceSynthetic Construct 60Met Ser Lys Gly
Glu Glu Leu Phe Thr Gly Val Val Pro Val Leu Val 1 5
10 15 Glu Leu Asp Gly Asp Val Asn Gly Gln
Lys Phe Ser Val Ser Gly Glu 20 25
30 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Asn Phe
Ile Cys 35 40 45
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50
55 60 Ser Tyr Gly Val Gln
Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70
75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu
Gly Tyr Val Gln Glu Arg 85 90
95 Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
Val 100 105 110 Lys
Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115
120 125 Asp Phe Lys Glu Asp Gly
Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135
140 Tyr Asn Ser His Asn Val Tyr Ile Met Gly Asp
Lys Pro Lys Asn Gly 145 150 155
160 Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175 Gln Leu
Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180
185 190 Val Leu Leu Pro Asp Asn His
Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200
205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Leu
Leu Glu Phe Val 210 215 220
Thr Ala Ala Arg Ile Thr His Gly Met Asp Glu Leu Tyr Lys 225
230 235 611350DNALeptosphaeria
nodorumCDS(1)..(1350)Native DNA sequence encoding Leptosphaeria
nodorum delta-9 fatty acid desaturase protein 61atg gcg gcc ttg gac agc
att cca gag gat aag gct acc tcg tcg aaa 48Met Ala Ala Leu Asp Ser
Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5
10 15 tcg act cat att caa tat caa gaa
gta act ttt cgg aac tgg tat aag 96Ser Thr His Ile Gln Tyr Gln Glu
Val Thr Phe Arg Asn Trp Tyr Lys 20 25
30 aag ata aat tgg ctc aac acg acg ctg gtg
gtg ctc ata ccc gct ctt 144Lys Ile Asn Trp Leu Asn Thr Thr Leu Val
Val Leu Ile Pro Ala Leu 35 40
45 gga ctc tac cta aca cgc acc acg cca ctt aca cga
cct acg ctc atc 192Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg
Pro Thr Leu Ile 50 55 60
tgg tcc gtc ctg tac tac ttc tgc aca gct ttc ggc atc aca
ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr
Gly Gly 65 70 75
80 tat cat cga cta tgg agt cat cgc agc tac tcc gct cgt cta ccg cta
288Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu
85 90 95
cgc tta ttc cta gcc ttc aca ggc gcc gga gcc atc caa ggt agt gct
336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala
100 105 110 cga
tgg tgg agc gca aat cac cgc gcc cac cac cga tgg acc gac aca 384Arg
Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr
115 120 125 atg aag gac
ccc tac tcc gtt atg cgc ggc cta tta ttc tcg cac atc 432Met Lys Asp
Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130
135 140 gga tgg atg gta ttg
aac agc gac ccc aaa gtc aaa ggc cga aca gac 480Gly Trp Met Val Leu
Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150
155 160 gtc agt gat ctc gac agc gac
ccc gtc gta gtc tgg cag cac aag cac 528Val Ser Asp Leu Asp Ser Asp
Pro Val Val Val Trp Gln His Lys His 165
170 175 tac ggc aag tgc ctg ctg ttc gcc gcg
tgg ata ttc ccc atg atc gta 576Tyr Gly Lys Cys Leu Leu Phe Ala Ala
Trp Ile Phe Pro Met Ile Val 180 185
190 gcc ggc ctc gga tgg gga gat tgg tgg gga ggc
ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly
Leu Val Tyr Ala Gly 195 200
205 atc att cga gcg tgt ttc gtc cag cag gcg aca ttt tgc
gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
ctc gcg cat tgg atc ggc gag cag ccg ttc gac gac aga cgc acg
cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr
Pro 225 230 235 240
cga gac cac gtt ttg aca gcg ttg gta acg atg gga gaa gga tat cat
768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His
245 250 255 aac
ttc cac cac gaa ttc cca agc gat tat cgc aac gcg atc atc tgg 816Asn
Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp
260 265 270 tac caa tac
gac cct acc aaa tgg ctc att tac ctc ttc tcc ctc ggc 864Tyr Gln Tyr
Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275
280 285 ccc ttc ccc ctc gca
tac tcg ctc aaa acc ttc cgg tcc aat gag att 912Pro Phe Pro Leu Ala
Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290
295 300 gaa aaa ggg cgg ttg caa caa
caa caa aaa gcc ctg gac aag aag cgc 960Glu Lys Gly Arg Leu Gln Gln
Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310
315 320 tca gga ctt gat tgg ggc cta ccc ctc
ttc caa ctc cct gtc ata tcg 1008Ser Gly Leu Asp Trp Gly Leu Pro Leu
Phe Gln Leu Pro Val Ile Ser 325 330
335 tgg gac gac ttc caa gcg cgt tgc aaa gag tcc ggc
gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly
Glu Met Leu Val 340 345
350 gct gtc gca ggt gtg att cac gac gtc agc cag ttt att gaa
gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu
Asp His 355 360 365
cct gga ggc agg agt ttg att cgg agt gcg gtg ggc aaa gat ggg aca
1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr
370 375 380 ggg atg
ttt aat gga ggc gta tat gag cac agt aat gcg gcg cat aat 1200Gly Met
Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385
390 395 400 ctg ttg tcg aca atg
agg gtg gga gtg ctt aga ggt ggg cag gag gtg 1248Leu Leu Ser Thr Met
Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405
410 415 gag gtg tgg aag aag cag aga gtg
gat gtt tta ggg aag agc gac att 1296Glu Val Trp Lys Lys Gln Arg Val
Asp Val Leu Gly Lys Ser Asp Ile 420 425
430 ttg aga cag gtt acg cgg gtg gag agg ttg gtt
gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val
Glu Gly Ala Val Ala 435 440
445 gcg tag
1350Ala
62449PRTLeptosphaeria nodorum 62Met Ala Ala Leu Asp Ser Ile
Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5
10 15 Ser Thr His Ile Gln Tyr Gln Glu Val Thr Phe
Arg Asn Trp Tyr Lys 20 25
30 Lys Ile Asn Trp Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala
Leu 35 40 45 Gly
Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50
55 60 Trp Ser Val Leu Tyr Tyr
Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65 70
75 80 Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser
Ala Arg Leu Pro Leu 85 90
95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala
100 105 110 Arg Trp
Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr 115
120 125 Met Lys Asp Pro Tyr Ser Val
Met Arg Gly Leu Leu Phe Ser His Ile 130 135
140 Gly Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys
Gly Arg Thr Asp 145 150 155
160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His
165 170 175 Tyr Gly Lys
Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180
185 190 Ala Gly Leu Gly Trp Gly Asp Trp
Trp Gly Gly Leu Val Tyr Ala Gly 195 200
205 Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr Pro 225
230 235 240 Arg Asp His Val
Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His 245
250 255 Asn Phe His His Glu Phe Pro Ser Asp
Tyr Arg Asn Ala Ile Ile Trp 260 265
270 Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser
Leu Gly 275 280 285
Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290
295 300 Glu Lys Gly Arg Leu
Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310
315 320 Ser Gly Leu Asp Trp Gly Leu Pro Leu Phe
Gln Leu Pro Val Ile Ser 325 330
335 Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly Glu Met Leu
Val 340 345 350 Ala
Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu Asp His 355
360 365 Pro Gly Gly Arg Ser Leu
Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370 375
380 Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser
Asn Ala Ala His Asn 385 390 395
400 Leu Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val
405 410 415 Glu Val
Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420
425 430 Leu Arg Gln Val Thr Arg Val
Glu Arg Leu Val Glu Gly Ala Val Ala 435 440
445 Ala 631350DNAArtificial SequenceSynthetic DNA
sequence encoding Leptosphaeria nodorum delta-9 fatty acid
desaturase protein using codons optimized for maize and Table 1 &
Table 2 sequences are maintainedCDS(1)..(1350) 63atg gca gcc ctt gac agc
atc cca gag gat aag gct acc tcg tct aaa 48Met Ala Ala Leu Asp Ser
Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1 5
10 15 tcg act cat att cag tac caa gaa
gtg act ttt cgg aac tgg tac aaa 96Ser Thr His Ile Gln Tyr Gln Glu
Val Thr Phe Arg Asn Trp Tyr Lys 20 25
30 aag ata aac tgg ctc aac acg acg ctg gtg
gtg ctc ata cca gct ctt 144Lys Ile Asn Trp Leu Asn Thr Thr Leu Val
Val Leu Ile Pro Ala Leu 35 40
45 ggt ctt tac cta aca agg acc acg cca ctt act agg
cca acg ctc atc 192Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg
Pro Thr Leu Ile 50 55 60
tgg tcc gtc ctg tac tac ttt tgc acc gct ttc ggc att acc
ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr
Gly Gly 65 70 75
80 tat cat aga cta tgg agt cat cgc agc tac tcc gct cgt cta ccg ctt
288Tyr His Arg Leu Trp Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu
85 90 95
cgc ttg ttc ctg gcc ttc act ggc gcc ggg gcc atc caa ggt tca gct
336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile Gln Gly Ser Ala
100 105 110 agg
tgg tgg agc gca aat cac cgc gcc cat cat agg tgg acc gac aca 384Arg
Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp Thr
115 120 125 atg aag gac
ccc tac tcc gtt atg cgc ggt cta tta ttc tcg cac atc 432Met Lys Asp
Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130
135 140 ggt tgg atg gtt cta
aac agc gac ccc aaa gtc aaa ggc cgc act gac 480Gly Trp Met Val Leu
Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150
155 160 gtc tca gac cta gat agc gac
ccc gtc gtt gtc tgg cag cac aag cac 528Val Ser Asp Leu Asp Ser Asp
Pro Val Val Val Trp Gln His Lys His 165
170 175 tac ggc aag tgc ctg cta ttt gcc gca
tgg ata ttc ccg atg atc gta 576Tyr Gly Lys Cys Leu Leu Phe Ala Ala
Trp Ile Phe Pro Met Ile Val 180 185
190 gcc ggc ctc gga tgg gga gat tgg tgg gga ggc
ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp Trp Trp Gly Gly
Leu Val Tyr Ala Gly 195 200
205 atc att agg gcg tgt ttc gtc cag caa gca acc ttt tgc
gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln Ala Thr Phe Cys
Val Asn Ser 210 215 220
ctc gcg cac tgg atc ggc gag cag ccg ttc gac gac aga cgc acc
cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp Arg Arg Thr
Pro 225 230 235 240
aga gac cac gtt ttg acc gcg ttg gtc act atg gga gaa ggt tat cac
768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His
245 250 255 aac
ttc cac cac gag ttc ccg tct gat tat agg aac gcg atc atc tgg 816Asn
Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp
260 265 270 tat cag tac
gac cct acc aaa tgg ctc ata tac ctc ttc tcc ctc ggc 864Tyr Gln Tyr
Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly 275
280 285 ccg ttc cca ctg gca
tac tcg ctc aaa acc ttc cgg tct aac gag atc 912Pro Phe Pro Leu Ala
Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290
295 300 gaa aag ggg cgg ttg caa caa
caa caa aag gcc ctg gat aag aag cgc 960Glu Lys Gly Arg Leu Gln Gln
Gln Gln Lys Ala Leu Asp Lys Lys Arg 305 310
315 320 tct ggc ctt gat tgg ggc ctg ccc ctc
ttc cag ctc cct gtg ata tct 1008Ser Gly Leu Asp Trp Gly Leu Pro Leu
Phe Gln Leu Pro Val Ile Ser 325 330
335 tgg gac gac ttc caa gcg cgt tgt aag gag tcc ggc
gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys Lys Glu Ser Gly
Glu Met Leu Val 340 345
350 gct gtc gcc ggt gtg att cac gac gtc tca cag ttt att gaa
gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu
Asp His 355 360 365
cct gga ggg agg agt ctg att cgg tct gcg gtg ggc aag gat ggg act
1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr
370 375 380 ggg atg
ttt aat gga ggc gtt tat gag cac agt aat gcg gcg cac aat 1200Gly Met
Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385
390 395 400 ctg ttg tca aca atg
agg gtg ggt gtg ctt aga ggt ggg caa gag gtg 1248Leu Leu Ser Thr Met
Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val 405
410 415 gag gtg tgg aag aag cag cgt gtg
gat gtt tta ggg aag agc gat atc 1296Glu Val Trp Lys Lys Gln Arg Val
Asp Val Leu Gly Lys Ser Asp Ile 420 425
430 ttg cgt caa gtt acg cgg gtg gag agg ctg gtt
gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val Glu Arg Leu Val
Glu Gly Ala Val Ala 435 440
445 gcc tag
1350Ala 64449PRTArtificial SequenceSynthetic Construct
64Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1
5 10 15 Ser Thr His Ile
Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20
25 30 Lys Ile Asn Trp Leu Asn Thr Thr Leu
Val Val Leu Ile Pro Ala Leu 35 40
45 Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr
Leu Ile 50 55 60
Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65
70 75 80 Tyr His Arg Leu Trp
Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85
90 95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly
Ala Ile Gln Gly Ser Ala 100 105
110 Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp
Thr 115 120 125 Met
Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130
135 140 Gly Trp Met Val Leu Asn
Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150
155 160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val
Trp Gln His Lys His 165 170
175 Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val
180 185 190 Ala Gly
Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195
200 205 Ile Ile Arg Ala Cys Phe Val
Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215
220 Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp
Arg Arg Thr Pro 225 230 235
240 Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His
245 250 255 Asn Phe His
His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260
265 270 Tyr Gln Tyr Asp Pro Thr Lys Trp
Leu Ile Tyr Leu Phe Ser Leu Gly 275 280
285 Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser
Asn Glu Ile 290 295 300
Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305
310 315 320 Ser Gly Leu Asp
Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325
330 335 Trp Asp Asp Phe Gln Ala Arg Cys Lys
Glu Ser Gly Glu Met Leu Val 340 345
350 Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu
Asp His 355 360 365
Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370
375 380 Gly Met Phe Asn Gly
Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390
395 400 Leu Leu Ser Thr Met Arg Val Gly Val Leu
Arg Gly Gly Gln Glu Val 405 410
415 Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp
Ile 420 425 430 Leu
Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435
440 445 Ala 651350DNAArtificial
SequenceSynthetic DNA sequence in accordance with the invention
encoding Leptosphaeria nodorum delta-9 fatty acid desaturase protein
using codons optimized for maize and with sequences identified in
Table 2 removed and Table 1 sequences are maintainedCDS(1)..(1350)
65atg gca gcc ctt gac agc atc cca gag gat aag gct acc tcg tct aaa
48Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1
5 10 15 tcg act
cat att cag tac caa gaa gtg act ttt cgg aac tgg tac aaa 96Ser Thr
His Ile Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys
20 25 30 aag ata aac tgg
ctc aac acg acg ctg gtg gtg ctc ata cca gct ctt 144Lys Ile Asn Trp
Leu Asn Thr Thr Leu Val Val Leu Ile Pro Ala Leu 35
40 45 ggt ctt tac cta aca agg
acc acg cca ctt act agg cca acg ctc atc 192Gly Leu Tyr Leu Thr Arg
Thr Thr Pro Leu Thr Arg Pro Thr Leu Ile 50 55
60 tgg tcc gtc ctg tac tac ttt tgc
acc gct ttc ggc att acc ggc gga 240Trp Ser Val Leu Tyr Tyr Phe Cys
Thr Ala Phe Gly Ile Thr Gly Gly 65 70
75 80 tat cat aga cta tgg agt cat cgc agc tac
tcc gct cgt cta ccg ctt 288Tyr His Arg Leu Trp Ser His Arg Ser Tyr
Ser Ala Arg Leu Pro Leu 85 90
95 cgc ttg ttc ctg gcc ttc act ggc gcc ggg gcc atc
caa ggt tca gct 336Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly Ala Ile
Gln Gly Ser Ala 100 105
110 agg tgg tgg agc gca aat cac cgc gcc cat cat agg tgg acc
gac aca 384Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr
Asp Thr 115 120 125
atg aag gac ccc tac tcc gtt atg cgc ggt ctg tta ttc tcg cac atc
432Met Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile
130 135 140 ggt
tgg atg gtt cta aac agc gac ccc aaa gtc aaa ggc cgc act gac 480Gly
Trp Met Val Leu Asn Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145
150 155 160 gtc tca gac
cta gat agc gac ccc gtc gtt gtc tgg cag cac aag cac 528Val Ser Asp
Leu Asp Ser Asp Pro Val Val Val Trp Gln His Lys His
165 170 175 tac ggc aag tgc ctg
cta ttt gcc gca tgg ata ttc ccg atg atc gta 576Tyr Gly Lys Cys Leu
Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val 180
185 190 gcc ggc ctc gga tgg gga gat
tgg tgg gga ggc ctt gtc tac gcc ggc 624Ala Gly Leu Gly Trp Gly Asp
Trp Trp Gly Gly Leu Val Tyr Ala Gly 195 200
205 atc att agg gcg tgt ttc gtc cag caa
gca acc ttt tgc gtg aac tct 672Ile Ile Arg Ala Cys Phe Val Gln Gln
Ala Thr Phe Cys Val Asn Ser 210 215
220 ctc gcg cac tgg atc ggc gag cag ccg ttc gac
gac aga cgc acc cct 720Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp
Asp Arg Arg Thr Pro 225 230 235
240 aga gac cac gtt ttg acc gcg ttg gtc act atg gga gaa
ggt tat cac 768Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu
Gly Tyr His 245 250
255 aac ttc cac cac gag ttc ccg tct gat tat agg aac gcg atc atc
tgg 816Asn Phe His His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile
Trp 260 265 270
tat cag tac gac cct acc aaa tgg ctc ata tac ctc ttc tcc ctc ggc
864Tyr Gln Tyr Asp Pro Thr Lys Trp Leu Ile Tyr Leu Phe Ser Leu Gly
275 280 285 ccg
ttc cca ctg gca tac tcg ctc aaa acc ttc cgg tct aac gag atc 912Pro
Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser Asn Glu Ile 290
295 300 gaa aag ggg
cgg ttg caa caa caa caa aag gcc ctg gat aag aag cgc 960Glu Lys Gly
Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305
310 315 320 tct ggc ctt gat tgg
ggc ctg ccc ctc ttc cag ctc cct gtg ata tct 1008Ser Gly Leu Asp Trp
Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325
330 335 tgg gac gac ttc caa gcg cgt tgt
aag gag tcc ggc gag atg ctg gtt 1056Trp Asp Asp Phe Gln Ala Arg Cys
Lys Glu Ser Gly Glu Met Leu Val 340 345
350 gct gtc gcc ggt gtg att cac gac gtc tca cag
ttc att gaa gat cac 1104Ala Val Ala Gly Val Ile His Asp Val Ser Gln
Phe Ile Glu Asp His 355 360
365 cct gga ggg agg agt ctg att cgg tct gcg gtg ggc aag
gat ggg act 1152Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys
Asp Gly Thr 370 375 380
ggg atg ttt aat gga ggc gtt tat gag cac agt aat gcg gcg cac aat
1200Gly Met Phe Asn Gly Gly Val Tyr Glu His Ser Asn Ala Ala His Asn
385 390 395 400 ctg
ttg tca aca atg agg gtg ggt gtg ctt aga ggt ggg caa gag gtg 1248Leu
Leu Ser Thr Met Arg Val Gly Val Leu Arg Gly Gly Gln Glu Val
405 410 415 gag gtg tgg aag
aag cag cgt gtg gat gta tta ggg aag agc gat atc 1296Glu Val Trp Lys
Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp Ile 420
425 430 ttg cgt caa gtt acg cgg gtg
gag agg ctg gtt gag ggg gct gtg gct 1344Leu Arg Gln Val Thr Arg Val
Glu Arg Leu Val Glu Gly Ala Val Ala 435 440
445 gcc tag
1350Ala
66449PRTArtificial SequenceSynthetic Construct
66Met Ala Ala Leu Asp Ser Ile Pro Glu Asp Lys Ala Thr Ser Ser Lys 1
5 10 15 Ser Thr His Ile
Gln Tyr Gln Glu Val Thr Phe Arg Asn Trp Tyr Lys 20
25 30 Lys Ile Asn Trp Leu Asn Thr Thr Leu
Val Val Leu Ile Pro Ala Leu 35 40
45 Gly Leu Tyr Leu Thr Arg Thr Thr Pro Leu Thr Arg Pro Thr
Leu Ile 50 55 60
Trp Ser Val Leu Tyr Tyr Phe Cys Thr Ala Phe Gly Ile Thr Gly Gly 65
70 75 80 Tyr His Arg Leu Trp
Ser His Arg Ser Tyr Ser Ala Arg Leu Pro Leu 85
90 95 Arg Leu Phe Leu Ala Phe Thr Gly Ala Gly
Ala Ile Gln Gly Ser Ala 100 105
110 Arg Trp Trp Ser Ala Asn His Arg Ala His His Arg Trp Thr Asp
Thr 115 120 125 Met
Lys Asp Pro Tyr Ser Val Met Arg Gly Leu Leu Phe Ser His Ile 130
135 140 Gly Trp Met Val Leu Asn
Ser Asp Pro Lys Val Lys Gly Arg Thr Asp 145 150
155 160 Val Ser Asp Leu Asp Ser Asp Pro Val Val Val
Trp Gln His Lys His 165 170
175 Tyr Gly Lys Cys Leu Leu Phe Ala Ala Trp Ile Phe Pro Met Ile Val
180 185 190 Ala Gly
Leu Gly Trp Gly Asp Trp Trp Gly Gly Leu Val Tyr Ala Gly 195
200 205 Ile Ile Arg Ala Cys Phe Val
Gln Gln Ala Thr Phe Cys Val Asn Ser 210 215
220 Leu Ala His Trp Ile Gly Glu Gln Pro Phe Asp Asp
Arg Arg Thr Pro 225 230 235
240 Arg Asp His Val Leu Thr Ala Leu Val Thr Met Gly Glu Gly Tyr His
245 250 255 Asn Phe His
His Glu Phe Pro Ser Asp Tyr Arg Asn Ala Ile Ile Trp 260
265 270 Tyr Gln Tyr Asp Pro Thr Lys Trp
Leu Ile Tyr Leu Phe Ser Leu Gly 275 280
285 Pro Phe Pro Leu Ala Tyr Ser Leu Lys Thr Phe Arg Ser
Asn Glu Ile 290 295 300
Glu Lys Gly Arg Leu Gln Gln Gln Gln Lys Ala Leu Asp Lys Lys Arg 305
310 315 320 Ser Gly Leu Asp
Trp Gly Leu Pro Leu Phe Gln Leu Pro Val Ile Ser 325
330 335 Trp Asp Asp Phe Gln Ala Arg Cys Lys
Glu Ser Gly Glu Met Leu Val 340 345
350 Ala Val Ala Gly Val Ile His Asp Val Ser Gln Phe Ile Glu
Asp His 355 360 365
Pro Gly Gly Arg Ser Leu Ile Arg Ser Ala Val Gly Lys Asp Gly Thr 370
375 380 Gly Met Phe Asn Gly
Gly Val Tyr Glu His Ser Asn Ala Ala His Asn 385 390
395 400 Leu Leu Ser Thr Met Arg Val Gly Val Leu
Arg Gly Gly Gln Glu Val 405 410
415 Glu Val Trp Lys Lys Gln Arg Val Asp Val Leu Gly Lys Ser Asp
Ile 420 425 430 Leu
Arg Gln Val Thr Arg Val Glu Arg Leu Val Glu Gly Ala Val Ala 435
440 445 Ala 67660DNAXerophyta
viscosaCDS(1)..(660)Native DNA sequence encoding Xerophyta viscosa
PER1 protein 67atg ccg ggg ctc acc att ggc gac acg atc ccc aac ctg gag
ctt gac 48Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu
Leu Asp 1 5 10 15
acc acc cag ggt agg atc aaa atc cac gat tac gtc ggc aac ggc tac
96Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr
20 25 30 gtc
atc ttg ttc tca cac cct gga gac ttc act cct gtc tgc acc acc 144Val
Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr
35 40 45 gaa ctt gga
aag atg gct gct tac gcc gac gag ttc agc aag cgc ggg 192Glu Leu Gly
Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50
55 60 gtt aag ctt ctt ggt
ctt tcc tgc gac gat gta cag agc cac aag gag 240Val Lys Leu Leu Gly
Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65 70
75 80 tgg atc aag gat atc gaa gcc
tat acg ccg gga tgt cac gta aaa tat 288Trp Ile Lys Asp Ile Glu Ala
Tyr Thr Pro Gly Cys His Val Lys Tyr 85
90 95 cct atc gcg gcg gac cca acc cgc gag
att atc cag cag cta aac atg 336Pro Ile Ala Ala Asp Pro Thr Arg Glu
Ile Ile Gln Gln Leu Asn Met 100 105
110 gta gac cca gac gag aca gag tcc agc aaa tgc
gcc gtg cct tcg cga 384Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys
Ala Val Pro Ser Arg 115 120
125 gct ctg cac atc att ggg ccc gac aag agg atc aag ctg
agt ttc ctg 432Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu
Ser Phe Leu 130 135 140
tac ccc gcg tcg acg ggg cga aac atg gat gag gtg ctg agg gca
gtg 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala
Val 145 150 155 160
gag tcg ctc cag cag gcg gca aag cac aag gtg gca acg ccg gcg aac
528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn
165 170 175 tgg
aag cct ggt gaa cct gtt gtg atc aag cct gat gtg tcc agc gag 576Trp
Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu
180 185 190 gag gcc aag
aag ctt ttc ccg cag ggt tat aaa agt gtt gat ctt cca 624Glu Ala Lys
Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195
200 205 tcc aag aag gat tac
ctt cgt ttt acg aac gtc tga 660Ser Lys Lys Asp Tyr
Leu Arg Phe Thr Asn Val 210
215 68219PRTXerophyta viscosa
68Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Leu Asp 1
5 10 15 Thr Thr Gln Gly
Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr 20
25 30 Val Ile Leu Phe Ser His Pro Gly Asp
Phe Thr Pro Val Cys Thr Thr 35 40
45 Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys
Arg Gly 50 55 60
Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65
70 75 80 Trp Ile Lys Asp Ile
Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85
90 95 Pro Ile Ala Ala Asp Pro Thr Arg Glu Ile
Ile Gln Gln Leu Asn Met 100 105
110 Val Asp Pro Asp Glu Thr Glu Ser Ser Lys Cys Ala Val Pro Ser
Arg 115 120 125 Ala
Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu Ser Phe Leu 130
135 140 Tyr Pro Ala Ser Thr Gly
Arg Asn Met Asp Glu Val Leu Arg Ala Val 145 150
155 160 Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val
Ala Thr Pro Ala Asn 165 170
175 Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu
180 185 190 Glu Ala
Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro 195
200 205 Ser Lys Lys Asp Tyr Leu Arg
Phe Thr Asn Val 210 215
69660DNAArtificial SequenceSynthetic DNA sequence encoding Xerophyta
viscosa PER1 protein using codons optimized for maize and Table 1 & Table
2 sequences are maintainedCDS(1)..(660) 69atg cct gga ttg act att ggt gac
aca att ccc aac ttg gag ctg gat 48Met Pro Gly Leu Thr Ile Gly Asp
Thr Ile Pro Asn Leu Glu Leu Asp 1 5
10 15 acg aca caa ggt cgc atc aag atc cac gac
tat gtc ggg aat gga tac 96Thr Thr Gln Gly Arg Ile Lys Ile His Asp
Tyr Val Gly Asn Gly Tyr 20 25
30 gtg att ctc ttc tca cat cct ggt gat ttc act ccg
gtg tgt acc acc 144Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro
Val Cys Thr Thr 35 40 45
gaa ttg ggc aag atg gct gct tat gcc gac gag ttc tct aag
cgt ggt 192Glu Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys
Arg Gly 50 55 60
gtg aag ctg ctt ggg ttg tcc tgt gat gat gtc caa tca cat aag gag
240Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu
65 70 75 80 tgg
atc aaa gac ata gag gct tac aca cct ggc tgt cac gta aaa tat 288Trp
Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr
85 90 95 ccg att gct
gct gat cca acc aga gaa atc ata cag cag ctg aac atg 336Pro Ile Ala
Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100
105 110 gtg gac cct gat gag
acg gaa agc tct aag tgc gct gtg cct tct agg 384Val Asp Pro Asp Glu
Thr Glu Ser Ser Lys Cys Ala Val Pro Ser Arg 115
120 125 gca ctt cac atc ata gga cca
gat aag agg atc aag ctg tcc ttc ctc 432Ala Leu His Ile Ile Gly Pro
Asp Lys Arg Ile Lys Leu Ser Phe Leu 130 135
140 tac cct gcc tct act ggt cgc aac atg
gac gaa gtt ctt aga gcc gtt 480Tyr Pro Ala Ser Thr Gly Arg Asn Met
Asp Glu Val Leu Arg Ala Val 145 150
155 160 gag tct ctt cag caa gca gct aaa cac aaa gtt
gca act cct gct aac 528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val
Ala Thr Pro Ala Asn 165 170
175 tgg aaa cct ggc gaa cca gtc gtc atc aaa cca gac gtc
agc tcc gag 576Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val
Ser Ser Glu 180 185 190
gag gcc aag aag ctc ttt cct caa ggt tat aaa agc gtt gat ttg
cct 624Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu
Pro 195 200 205
tca aag aag gac tac ttg agg ttc acc aat gtt tga
660Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val
210 215 70219PRTArtificial
SequenceSynthetic Construct 70Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro
Asn Leu Glu Leu Asp 1 5 10
15 Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr
20 25 30 Val Ile
Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35
40 45 Glu Leu Gly Lys Met Ala Ala
Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55
60 Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln
Ser His Lys Glu 65 70 75
80 Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr
85 90 95 Pro Ile Ala
Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100
105 110 Val Asp Pro Asp Glu Thr Glu Ser
Ser Lys Cys Ala Val Pro Ser Arg 115 120
125 Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu
Ser Phe Leu 130 135 140
Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145
150 155 160 Glu Ser Leu Gln
Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165
170 175 Trp Lys Pro Gly Glu Pro Val Val Ile
Lys Pro Asp Val Ser Ser Glu 180 185
190 Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp
Leu Pro 195 200 205
Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215
71660DNAArtificial SequenceSynthetic DNA sequence in
accordance with the invention encoding Xerophyta viscosa PER1
protein using codons optimized for maize and with sequences
identified in Table 2 removed and Table 1 sequences are
maintainedCDS(1)..(660) 71atg cct gga ttg act att ggt gac aca att ccc aac
ttg gag ctg gat 48Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro Asn
Leu Glu Leu Asp 1 5 10
15 acg aca caa ggt cgc atc aag atc cac gac tat gtc ggg aat
gga tac 96Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn
Gly Tyr 20 25 30
gtg att ctc ttc tca cat cct ggt gat ttc act ccg gtg tgt acc acc
144Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr
35 40 45 gaa
ttg ggc aag atg gct gct tat gcc gac gag ttc tct aag cgt ggt 192Glu
Leu Gly Lys Met Ala Ala Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50
55 60 gtg aag ctg
ctt ggg ttg tcc tgt gat gat gtc caa tca cat aag gag 240Val Lys Leu
Leu Gly Leu Ser Cys Asp Asp Val Gln Ser His Lys Glu 65
70 75 80 tgg atc aaa gac ata
gag gct tac aca cct ggc tgt cac gta aaa tat 288Trp Ile Lys Asp Ile
Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr 85
90 95 ccg att gct gct gat cca acc
aga gaa atc ata cag cag ctg aac atg 336Pro Ile Ala Ala Asp Pro Thr
Arg Glu Ile Ile Gln Gln Leu Asn Met 100
105 110 gtg gac cct gat gag acg gaa agc tct
aag tgc gct gtg cct tct agg 384Val Asp Pro Asp Glu Thr Glu Ser Ser
Lys Cys Ala Val Pro Ser Arg 115 120
125 gca ctt cac atc ata gga cca gat aag agg atc
aag ctg tcc ttc ctc 432Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile
Lys Leu Ser Phe Leu 130 135 140
tac cct gcc tct act ggt cgc aac atg gac gaa gtt ctt
aga gcc gtt 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu
Arg Ala Val 145 150 155
160 gag tct ctt cag caa gca gct aaa cac aaa gtt gca act cct gct
aac 528Glu Ser Leu Gln Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala
Asn 165 170 175
tgg aaa cct ggc gaa cca gtc gtc atc aaa cca gac gtc agc tcc gag
576Trp Lys Pro Gly Glu Pro Val Val Ile Lys Pro Asp Val Ser Ser Glu
180 185 190 gag
gcc aag aag ctc ttt cct caa ggt tat aaa agc gtt gat ttg cct 624Glu
Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp Leu Pro
195 200 205 tca aag aag
gac tac ttg agg ttc acc aat gtt tga 660Ser Lys Lys
Asp Tyr Leu Arg Phe Thr Asn Val 210
215 72219PRTArtificial
SequenceSynthetic Construct 72Met Pro Gly Leu Thr Ile Gly Asp Thr Ile Pro
Asn Leu Glu Leu Asp 1 5 10
15 Thr Thr Gln Gly Arg Ile Lys Ile His Asp Tyr Val Gly Asn Gly Tyr
20 25 30 Val Ile
Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35
40 45 Glu Leu Gly Lys Met Ala Ala
Tyr Ala Asp Glu Phe Ser Lys Arg Gly 50 55
60 Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln
Ser His Lys Glu 65 70 75
80 Trp Ile Lys Asp Ile Glu Ala Tyr Thr Pro Gly Cys His Val Lys Tyr
85 90 95 Pro Ile Ala
Ala Asp Pro Thr Arg Glu Ile Ile Gln Gln Leu Asn Met 100
105 110 Val Asp Pro Asp Glu Thr Glu Ser
Ser Lys Cys Ala Val Pro Ser Arg 115 120
125 Ala Leu His Ile Ile Gly Pro Asp Lys Arg Ile Lys Leu
Ser Phe Leu 130 135 140
Tyr Pro Ala Ser Thr Gly Arg Asn Met Asp Glu Val Leu Arg Ala Val 145
150 155 160 Glu Ser Leu Gln
Gln Ala Ala Lys His Lys Val Ala Thr Pro Ala Asn 165
170 175 Trp Lys Pro Gly Glu Pro Val Val Ile
Lys Pro Asp Val Ser Ser Glu 180 185
190 Glu Ala Lys Lys Leu Phe Pro Gln Gly Tyr Lys Ser Val Asp
Leu Pro 195 200 205
Ser Lys Lys Asp Tyr Leu Arg Phe Thr Asn Val 210 215
* * * * *
