U.S. patent application number 15/518677 was filed with the patent office on 2017-08-10 for insecticidal polypeptides having improved activity spectrum and uses thereof.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The applicant listed for this patent is PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to MICHI IZUMI WILCOXON, TAKASHI YAMAMOTO.
Application Number | 20170226164 15/518677 |
Document ID | / |
Family ID | 55747257 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226164 |
Kind Code |
A1 |
IZUMI WILCOXON; MICHI ; et
al. |
August 10, 2017 |
INSECTICIDAL POLYPEPTIDES HAVING IMPROVED ACTIVITY SPECTRUM AND
USES THEREOF
Abstract
The disclosure provides nucleic acids, and variants and
fragments thereof, derived from strains of Bacillus thuringiensis
encoding variant polypeptides having increased pesticidal activity
against insect pests, including Lepidoptera and Coleopteran.
Particular embodiments of the disclosure provide isolated nucleic
acids encoding pesticidal proteins, pesticidal compositions, DNA
constructs, and transformed microorganisms and plants comprising a
nucleic acid of the embodiments. These compositions find use in
methods for controlling pests, especially plant pests.
Inventors: |
IZUMI WILCOXON; MICHI;
(SUNNYVALE, CA) ; YAMAMOTO; TAKASHI; (DUBLIN,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC. |
JOHNSTON |
IA |
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
JOHNSTON
IA
|
Family ID: |
55747257 |
Appl. No.: |
15/518677 |
Filed: |
October 14, 2015 |
PCT Filed: |
October 14, 2015 |
PCT NO: |
PCT/US15/55491 |
371 Date: |
April 12, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62064877 |
Oct 16, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 40/162 20180101;
C12N 15/8286 20130101; C07K 14/325 20130101; Y02A 40/146 20180101;
A01N 63/10 20200101; A01N 37/46 20130101; A01N 65/22 20130101; A01N
65/44 20130101; A01N 65/44 20130101; A01N 37/46 20130101; A01N
57/16 20130101; A01N 63/10 20200101; A01N 65/22 20130101; A01N
37/46 20130101; A01N 57/16 20130101; A01N 63/10 20200101; A01N
65/44 20130101; A01N 37/46 20130101; A01N 57/16 20130101; A01N
63/10 20200101; A01N 65/22 20130101; A01N 37/46 20130101; A01N
57/16 20130101; A01N 63/10 20200101 |
International
Class: |
C07K 14/325 20060101
C07K014/325; C12N 15/82 20060101 C12N015/82; A01N 63/02 20060101
A01N063/02 |
Claims
1. A variant Cry1B polypeptide comprising an amino acid sequence
having at least one amino acid substitution compared to the
corresponding reference Cry1B polypeptide, wherein the variant
Cry1B polypeptide has increased insecticidal activity against corn
earworm and/or fall armyworm compared to the corresponding
reference Cry1B polypeptide.
2. The variant Cry1B polypeptide of claim 1, wherein the
corresponding reference Cry1B polypeptide is selected from: a) a
polypeptide comprising the amino acid sequence of SEQ ID NO: 1; b)
a polypeptide comprising the amino acid sequence of SEQ ID NO: 47;
c) a polypeptide comprising the amino acid sequence of SEQ ID NO:
52; and d) a polypeptide comprising the amino acid sequence of SEQ
ID NO: 54.
3. (canceled)
4. (canceled)
5. (canceled)
6. The variant Cry1B polypeptide of claim 1, wherein the
corresponding reference Cry1B polypeptide comprises the amino
sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35,
SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ
ID NO: 45.
7. The variant Cry1B polypeptide of claim 1, wherein the
corresponding reference Cry1B polypeptide comprises a Cry1Be type
Domain I and a Cry1Ah type Domain III.
8. The variant Cry1B polypeptide of claim 7, wherein Cry1Be type
Domain I is selected from; a) a Cry1Be type Domain I having at
least 90% identity to amino acids 36-276 of SEQ ID NO: 58; and b) a
Cry1Be type Domain I having at least 90% identity to amino acids
35-276 of SEQ ID NO: 47.
9. (canceled)
10. The variant Cry1B polypeptide of claim 7 or 8, wherein the
Cry1Ah type Domain III is selected from: a) a Cry1Ah type Domain
III having at least 80% sequence identity to amino acids 483 to 643
of SEQ ID NO: 61; and b) a Cry1Ah type Domain III having at least
80% sequence identity to amino acids 494 to 655 of SEQ ID NO:
47.
11. (canceled)
12. The variant Cry1B polypeptide of claim 1, wherein the
corresponding reference Cry1B polypeptide comprises a Cry1Ba type
Domain I and Domain II selected from: a) a Cry1Ba type Domain I and
Domain II having at least 80% sequence identity to amino acids 30
to 489 of SEQ ID NO: 55; b) a Cry1Be type Domain I and Domain II
having at least 80% sequence identity to amino acids 35 to 494 of
SEQ ID NO: 58; and c) a Cry1Be type Domain I and Domain II having
at least 80% sequence identity to amino acids 35 to 493 of SEQ ID
NO: 47.
13. (canceled)
14. (canceled)
15. The variant Cry1B polypeptide of claim 12, wherein the
corresponding reference Cry1B polypeptide further comprises a
Cry1Ah type Domain III selected from: a) a Cry1Ah type Domain III
having at least 80% sequence identity to amino acids 483 to 643 of
SEQ ID NO: 61; and b) a Cry1Ah type Domain III having at least 80%
sequence identity to amino acids 494 to 655 of SEQ ID NO: 47.
16. (canceled)
17. The variant Cry1B polypeptide of claim 1, wherein the variant
Cry1B polypeptide comprises an amino acid substitution at position
50, 57, 65, 67, 68, 70, 71, 73, 74, 76, 77, 79, 80, 82, 83, 87, 91,
92, 93, 94, 95, 106, 108, 109, 110, 111, 112, 113, 114, 115, 118,
119, 122, 123, 125, 129, 136, 140, 143, 144, 145, 146, 147, 148,
149, 158, 159, 160, 166, 167, 173, 177, 178, 179, 180, 201, 206,
209, 210, 211, 213, 214, 218, 219, 221, 222, 225, 226, 230, 233,
234, 236, 240, 241, 242, 243, 245, 246, 247, 248, 252, 277, 280,
281, 303, 306, 360, 362, 367, 406, 407, 418, 425, 427, 429, 431,
435, 439, 447, 473, 476, 477, 478, 479, 490, 495, 499, 502, 509,
512, 513, 515, 517, 518, 520, 521, 526, 532, 534, 535, 537, 538,
541, 545, 547, 551, 552, 553, 554, 555, 556, 557, 558, 559, 563,
564, 565, 568, 569, 570, 571, 572, 573, 574, 577, 581, 582, 583,
584, 585, 586, 587, 590, 591, 592, 593, 595, 596, 598, 599, 601,
602, 603, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615,
618, 624, 626, 628, 629, 636, 641, 643, 645, or 646 corresponding
to the amino acid sequence of SEQ ID NO: 47.
18. The variant Cry1B polypeptide of claim 17, wherein the amino
acid substitution at position 50 is selected from R, I, D, A, H, V,
S, F, V, K, and N; at position 57 is selected from V, R, L, N, G,
and D; at position 65 is selected from Q, A, S, and G; at position
67 is selected from M, F, and I; at position 68 is selected from A,
R, and F; at position 70 is selected from E, W, and H; at position
71 is S; at position 73 is selected from S and G; at position 74 is
selected from I, E, S, R, V, and D; at position 76 is selected from
T, S, Y, V, D, and R; at position 77 is selected from N, D, G, L,
I, H, P, A, T, M, C, and S; at position 79 is selected from S, V,
T, L, R, I, P, N, Q, and K; at position 80 is selected from Q, K,
G, E, R, M, N, C, W, Y, and D; at position 82 is F; at position 83
is selected from E, D, G, A, K, H, R, Y, and L; at position 87 is
selected from D, K, N, C, W, and H; at position 91 is selected from
S, Y, T, and D; at position 92 is selected from E, G, F, V, L, and
T; at position 93 is selected from H, D, and I; at position 94 is
selected from L, H, T, and S; at position 95 is selected from G, Q,
V, and F; at position 106 is selected from I, A, F, G, H, C, K, V,
R, and S; at position 108 is selected from L, M, and T; at position
109 is selected from S, V, and N; at position 110 is selected from
T, R, V, F, and H; at position 111 is selected from H, L, S, M, R,
G, A, Q, N, K, Y, and E; at position 112 is L; at position 113 is
selected from L, V, S, N, and K; at position 114 is selected from
L, T, M, H, F, I, Y, A, S, V, E, and D; at position 115 is P; at
position 118 is selected from V, T, E, D, F, V, and G; at position
119 is selected from A, M, S, K, H, E, R, and V; at position 122 is
selected from R, I, F, N, G, and T; at position 123 is K; at
position 125 is selected from N, R, and E; at position 129 is
selected from K, W, L, P, and V; at position 136 is selected from
I, F, and I; at position 140 is E; at position 143 is selected from
S, R, G, Y, M, Q, L, W, T, A, N, and P; at position 144 is selected
from M, A, and T; at position 145 is selected from N, P, A, L, and
S; at position 146 is selected from W, T, H, and V; at position 147
is selected from V, R, D, and S; at position 148 is selected from
F, W, P, N, and L; at position 149 is selected from V, A, S, and L;
at position 158 is F; at position 159 is V; at position 160 is V;
at position 166 is selected from V, E, C, I, and T; at position 167
is selected from T, M, Q, L, and A; at position 173 is selected
from F, and T; at position 177 is selected from C, S, T, and P; at
position 178 is K; at position 179 is selected from I, and L; at
position 180 is selected from A, S, and L, M; at position 201 is V;
at position 206 is selected from L, I, T, and W; at position 209 is
selected from E, R, D, L, V, and C; at position 210 is selected
from P, T, I, and R; at position 211 is selected from I, R, G, T,
P, and L; at position 213 is selected from V, T, L, M, Q, N, and G;
at position 214 is W; at position 218 is selected from T, A, H, S,
I, V, Y, W, and D; at position 219 is N; at position 221 is
selected from L, Y, V, K, I, D, G, H, W, R, T, and F; at position
222 is selected from G, M, K, T, D, and I; at position 225 is
selected from V, Q, M, F, L, G, I, Y, C, N, and T; at position 226
is selected from D, S, V, C, Y, R, and A; at position 230 is
selected from A, L, and S; at position 233 is selected from K, D,
Q, G, I, A, and Y; at position 234 is selected from V, M, L, I, A,
R, F, Y, and S; at position 236 is selected from E, K, S, T, and L;
at position 240 is selected from Y, A, M, S, T, G, K, F, L, R, W,
and C; at position 241 is selected from S, I, W, M, K, Y, V, L, and
C; at position 242 is selected from P, and V; at position 243 is
selected from M, V, T, C, K, I, S, and Q; at position 245 is
selected from Q, Y, K, G, A, I, W, H, S, M, D, N, V, R, and F; at
position 246 is selected from T, S, G, and Q; at position 247 is
selected from E, S, G, and P; at position 248 is selected from S,
N, T, L, Y, V, R, and F; at position 252 is selected from N, A, and
F; at position 277 is selected from Q, G, and V; at position 280 is
selected from H, C, and T; at position 281 is selected from Q, M,
R, K, S, H, and A; at position 303 is selected from N, and P; at
position 306 is G; at position 360 is selected from S, N, T, Y, and
M; at position 362 is selected from Y, H, W, K, I, D, V, A, L, G,
and E; at position 367 is selected from H, Q, N, W, T, L, Y, I, and
A; at position 406 is M; at position 407 is W; at position 418 is
selected from K, and T; at position 425 is selected from P, and G;
at position 427 is Y; at position 429 is I; at position 431 is
selected from L, H, G, and A; at position 435 is selected from Y,
H, and L; at position 439 is selected from M, and Q; at position
447 is selected from N, V, I, S, L, A, E, and M; at position 473 is
selected from T, G, A, S, M, N, K, D, and Y; at position 476 is
selected from Y, H, G, L, S, F, and M; at position 477 is selected
from S, and A; at position 478 is selected from G, and K; at
position 479 is V; at position 490 is Q; at position 495 is N; at
position 499 is selected from R, S, G, M, C, V, P, and W; at
position 502 is selected from K, V, A, T, N, E, L, Q, P, H, R, F,
S, and Y; at position 509 is T; at position 512 is selected from A,
Y, P, M, R, K, G, S, Q, I, and W; at position 513 is selected from
G, V, P, L, H, and C; at position 515 is H; at position 517 is
selected from A, H, and S; at position 518 is selected from D, A,
Y, K, V, L, G, H, E, R, T, and C; at position 520 is selected from
V, R, Y, C, K, M, E, L, F, S, and A; at position 521 is selected
from G, L, V, A, D, I, Q, F, P, N, and M; at position 526 is L; at
position 532 is selected from K, C, W, S, L, V, H, and G; at
position 534 is selected from S, Y, Q, W, E, H, D, and L; at
position 535 is selected from M, Q, E, K, F, D, R, L, A, P, S, and
I; at position 537 is selected from W, E, F, A, K, S, Q, Y, R, C,
D, V, N, H, and T; at position 538 is selected from M, G, I, C, H,
T, S, F, A, Y, V, W, L, Q, I, D, E, and K; at position 541 is
selected from Y, N, W, and F; at position 545 is selected from F,
and L; at position 547 is selected from A, S, G, I, M, and Q; at
position 551 is selected from H, C, R, A, S, D, and Y; at position
552 is selected from T, V, and W; at position 553 is selected from
Q, D, R, E, A, F, L, P, G, W, S, and T; at position 554 is selected
from Y, R, D, H, N, and G; at position 555 is selected from V, M,
I, and W; at position 556 is selected from A, W, G, D, C, and P; at
position 557 is selected from I, R, G, S, Q, M, V, A, and C; at
position 558 is selected from Y, K, T, L, N, G, S, E, I, D, F, P,
V, M, and H; at position 559 is W; at position 563 is selected from
N, L, I, and A; at position 564 is selected from H, V, W, I, K, C,
S, and A; at position 565 is F; at position 568 is selected from C,
A, E, F, R, G, L, S, W, and N; at position 569 is selected from I,
M, G, and S; at position 570 is selected from M, F, W, and T; at
position 571 is selected from G, V, T, C, and L; at position 572 is
selected from H, P, R, I, K, F, S, A, V, W, and M; at position 573
is selected from A, T, and G; at position 574 is R; at position 577
is selected from R, F, K, M, V, A, T, H, G, and I; at position 581
is selected from S, and K; at position 582 is V; at position 583 is
S; at position 584 is R; at position 585 is selected from R, T, K,
H, Q, L, W, N, M, F, and I; at position 586 is selected from M, Y,
P, A, S, K, R, F, G, V, Q, N, L, W, and T; at position 587 is
selected from H, C, N, S, D, R, A, T, K, E, W, L, Y, F, and Q; at
position 590 is selected from A, D, F, S, and G; at position 591 is
selected from H, V, N, T, D, R, S, K, C, E, W, L, Y, F, P, and Q;
at position 592 is selected from Q, M, A, Y, N, K, P, S, D, I, G,
F, V, and W; at position 593 is selected from Y, G, R, and V; at
position 595 is selected from R, G, H, N, V, F, K, T, Y, I, M, A,
and P; at position 596 is selected from V, T, I, S, G, L, W, Y, H,
P, and D; at position 598 is selected from V, G, D, and I; at
position 599 is selected from C, Q, L, Y, T, V, A, and P; at
position 601 is selected from Y, F, V, G, M, and E; at position 602
is M; at position 603 is selected from M, A, Y, R, S, L, W, D, and
T; at position 605 is selected from S, W, R, M, A, I, C, V, K, D,
Y, N, Q, G, E, and P; at position 606 is selected from R, H, K, F,
Q, W, G, Y, M, T, A, I, L, and N; at position 607 is selected from
R, C, T, I, Q, G, D, E, and V; at position 608 is selected from R,
S, V, L, F, G, Y, A, K, W, and Q; at position 609 is selected from
G, P, L, R, S, V, F, and I; at position 610 is selected from G, F,
P, and L; at position 611 is selected from L, K, G, W, and V; at
position 612 is selected from F, H, G, E, N, D, and P; at position
613 is selected from M, T, W, V, N, R, and Y; at position 614 is
selected from M, S, L, H, V, R, G, Y, and D; at position 615 is
selected from V, Q, G, K, M, R, C, and L; at position 618 is
selected from N, H, W, R, G, L, D, and T; at position 624 is
selected from A, and M; at position 626 is selected from K, G, R,
T, H, A, N, I, Y, Q, P, and S; at position 628 is selected from F,
K, Q, S, R, G, L, I, and D; at position 629 is selected from L, V,
K, H, M, S, R, C, Q, E, T, P, and A; at position 636 is selected
from A, and C; at position 641 is selected from P, H, A, L, Q, Y,
E, I, S, V, D, and G; at position 643 is selected from L, A, Q, H,
S, D, M, C, and R; at position 645 is selected from T, M, L, Y, A,
N, V, P, I, W, C, S; and at position 646 is selected from S, Y, D,
E, M, F, H, V, W, and I.
19. The variant Cry1B polypeptide of claim 1, wherein the variant
Cry1B polypeptide is selected from: a) a variant Cry1B polypeptide
having has at least 80% identity to SEQ ID NO: 1; b) a variant
Cry1B polypeptide having at least 80% identity to SEQ ID NO: 47; c)
a variant Cry1B polypeptide having at least 80% identity to SEQ ID
NO: 52; and d) a variant Cry1B polypeptide having at least 80%
identity to SEQ ID NO: 54.
20. (canceled)
21. (canceled)
22. (canceled)
23. The variant Cry1B polypeptide of claim 1, wherein the variant
Cry1B polypeptide comprises an amino acid sequence having at least
95% sequence identity to SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43
or SEQ ID NO: 45.
24. The variant Cry1B polypeptide of claim 1, wherein the variant
Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45.
25. The variant Cry1B polypeptide of claim 1, wherein the variant
Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45.
26. The variant Cry1B polypeptide of claim 1 wherein the increased
insecticidal activity is against corn earworm.
27. The variant Cry1B polypeptide of claim 26, wherein the
increased insecticidal activity is quantified as a Mean FAE
index.
28. The variant Cry1B polypeptide of claim 1, wherein the increased
insecticidal activity is against fall armyworm.
29. The variant Cry1B polypeptide of claim 28, wherein the variant
Cry1B polypeptide comprises the amino acid sequence of SEQ ID NO:
23, SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29.
30. A recombinant polynucleotide encoding the variant Cry1B
polypeptide of claim 1.
31. The recombinant polynucleotide of claim 30, wherein the nucleic
acid sequence has been optimized for expression in a plant.
32. The recombinant polynucleotide of claim 31, wherein the nucleic
acid sequence is a synthetic nucleotide sequence having plant
preferred codons that have been designed for expression in a
plant.
33. The recombinant polynucleotide of claim 32, wherein the nucleic
acid sequence has been optimized for expression in maize or
soybean.
34. The recombinant polynucleotide of claim 30, wherein the
polynucleotide comprises a nucleic acid sequence of SEQ ID NO: 4,
SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46.
35. A DNA construct comprising a polynucleotide of claim 30
operably linked to a heterologous regulatory element.
36. A host cell comprising the DNA construct of claim 35.
37. The host cell of claim 36, wherein the host cell is a bacterial
cell.
38. The host cell of claim 37, wherein the host cell is a plant
cell.
39. The host cell of claim 38, wherein the host cell is a soybean
or maize cell.
40. A transgenic plant comprising the DNA construct of claim
30.
41. The transgenic plant of claim 40, wherein said plant is
selected from the group consisting of maize, sorghum, wheat,
cabbage, sunflower, tomato, a crucifer species, a pepper species,
potato, cotton, rice, soybean, sugar beet, sugarcane, tobacco,
barley, and oilseed rape.
42. A seed comprising the DNA construct of claim 35.
43. A composition comprising the variant Cry1B polypeptide of claim
1.
44. The composition of claim 43, wherein the composition comprises
from 1% to 99% by weight of the variant Cry1B polypeptide.
45. A method for controlling a Lepidopteran pest population
comprising contacting said population with a pesticidally-effective
amount of the variant Cry1B polypeptide of claim 1.
46. A method for killing a Lepidopteran pest comprising contacting
said pest with, or feeding to said pest, a pesticidally-effective
amount of the variant Cry1B polypeptide of claim 1.
47. A method for producing a polypeptide with pesticidal activity,
comprising culturing the host cell of claim 36 under conditions in
which the polynucleotide encoding the polypeptide is expressed.
48. A plant or plant cell having stably incorporated into its
genome the DNA construct of claim 35.
49. A method of protecting a plant from an insect pest, comprising
introducing into said plant the DNA construct of claim 35.
50. The method of claim 49, wherein the variant Cry1B polypeptide
has increased insecticidal activity against corn earworm and/or
fall armyworm compared to the polypeptide of SEQ ID NO: 47.
51. The method of claim 49 or 50, wherein the insect pest is
resistant to a non-Cry1B insecticidal polypeptide.
52. The method of claim 49, wherein the insect pest is corn
earworm, fall armyworm or European corn borer.
Description
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0001] A sequence listing having the file name
"5409WOPCT_SequenceListing.txt" created on Sep. 24, 2015 and having
a size of 267 kilobytes is filed in computer readable form
concurrently with the specification. The sequence listing is part
of the specification and is herein incorporated by reference in its
entirety.
FIELD
[0002] The present disclosure relates to recombinant nucleic acids
that encode pesticidal polypeptides having insecticidal activity
against corn earworm and/or fall armyworm and/or an improved
spectrum of pesticidal activity against insect pests. Compositions
and methods of the disclosure utilize the disclosed nucleic acids,
and their encoded pesticidal polypeptides, to control plant
pests.
BACKGROUND
[0003] Insect pests are a major factor in the loss of the world's
agricultural crops. For example, armyworm feeding, black cutworm
damage, or European corn borer damage can be economically
devastating to agricultural producers. Insect pest-related crop
loss from European corn borer attacks on field and sweet corn alone
has reached about one billion dollars a year in damage and control
expenses.
[0004] Traditionally, the primary method for impacting insect pest
populations is the application of broad-spectrum chemical
insecticides. However, consumers and government regulators alike
are becoming increasingly concerned with the environmental hazards
associated with the production and use of synthetic chemical
pesticides. Because of such concerns, regulators have banned or
limited the use of some of the more hazardous pesticides. Thus,
there is substantial interest in developing alternative
pesticides.
[0005] Biological control of insect pests of agricultural
significance using a microbial agent, such as fungi, bacteria, or
another species of insect affords an environmentally friendly and
commercially attractive alternative to synthetic chemical
pesticides. Generally speaking, the use of biopesticides presents a
lower risk of pollution and environmental hazards, and
biopesticides provide greater target specificity than is
characteristic of traditional broad-spectrum chemical insecticides.
In addition, biopesticides often cost less to produce and thus
improve economic yield for a wide variety of crops.
[0006] Certain species of microorganisms of the genus Bacillus are
known to possess pesticidal activity against a broad range of
insect pests including Lepidoptera, Diptera, Coleoptera, Hemiptera,
and others. Bacillus thuringiensis (Bt) and Bacillus papilliae are
among the most successful biocontrol agents discovered to date.
Insect pathogenicity has also been attributed to strains of B.
larvae, B. lentimorbus, B. sphaericus (Harwook, ed., ((1989)
Bacillus (Plenum Press), 306), and B. cereus (WO 96/10083).
Pesticidal activity appears to be concentrated in parasporal
crystalline protein inclusions, although pesticidal proteins have
also been isolated from the vegetative growth stage of Bacillus.
Several genes encoding these pesticidal proteins have been isolated
and characterized (see, for example, U.S. Pat. Nos. 5,366,892 and
5,840,868).
[0007] Microbial insecticides, particularly those obtained from
Bacillus strains, have played an important role in agriculture as
alternatives to chemical pest control. Recently, agricultural
scientists have developed crop plants with enhanced insect
resistance by genetically engineering crop plants to produce
pesticidal proteins from Bacillus. For example, corn and cotton
plants have been genetically engineered to produce pesticidal
proteins isolated from strains of Bt (see, e.g., Aronson (2002)
Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998) Microbiol
Mol Biol Rev. 62(3):775-806). These genetically engineered crops
are now widely used in American agriculture and have provided the
farmer with an environmentally friendly alternative to traditional
insect-control methods. In addition, potatoes genetically
engineered to contain pesticidal Cry toxins have been sold to the
American farmer. While they have proven to be very successful
commercially, these genetically engineered, insect-resistant crop
plants provide resistance to only a narrow range of the
economically important insect pests.
[0008] Accordingly, there remains a need for new Bt toxins with an
improved spectrum of insecticidal activity against insect pests,
e.g., toxins which are improved active against insects from the
order Lepidoptera and/or Coleoptera. In addition, there remains a
need for biopesticides having activity against a variety of insect
pests and for biopesticides which have improved insecticidal
activity.
SUMMARY
[0009] Compositions and methods are provided for impacting insect
pests. More specifically, the embodiments of the present disclosure
relate to methods of impacting insects utilizing nucleotide
sequences encoding insecticidal peptides to produce transformed
microorganisms and plants that express an insecticidal polypeptide
of the embodiments. In some embodiments, the nucleotide sequences
encode polypeptides that are pesticidal for at least one insect
belonging to the order Lepidoptera.
[0010] In some aspects nucleic acid molecules and fragments and
variants thereof are provided, which encode polypeptides that
possess pesticidal activity against insect pests (e.g. SEQ ID NO:
4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID
NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40,
SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46, and encoding the
polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID
NO: 45, respectively). The wild-type (e.g., naturally occurring)
nucleotide sequence of the embodiments, which was obtained from Bt,
encodes an insecticidal peptide. The embodiments further provide
fragments and variants of the disclosed nucleotide sequence that
encode biologically active (e.g., insecticidal) polypeptides.
[0011] In another aspect variant Cry1B polypeptides are provided,
encoded by a modified (e.g., mutagenized or manipulated) nucleic
acid molecule of the embodiments. In particular examples,
pesticidal proteins of the embodiments include fragments of
full-length proteins and polypeptides that are produced from
mutagenized nucleic acids designed to introduce particular amino
acid sequences into the polypeptides of the embodiments. In
particular embodiments, the polypeptides have enhanced pesticidal
activity relative to the activity of the naturally occurring
polypeptide from which they are derived.
[0012] In another aspect the nucleic acids of the embodiments can
also be used to produce transgenic (e.g., transformed) monocot or
dicot plants that are characterized by genomes that comprise at
least one stably incorporated nucleotide construct comprising a
coding sequence of the embodiments operably linked to a promoter
that drives expression of the encoded pesticidal polypeptide.
Accordingly, transformed plant cells, plant tissues, plants, and
seeds thereof are also provided.
[0013] In another aspect transformed plant can be produced using a
nucleic acid that has been optimized for increased expression in a
host plant. For example, one of the pesticidal polypeptides of the
embodiments can be back-translated to produce a nucleic acid
comprising codons optimized for expression in a particular host,
for example a crop plant such as a corn (Zea mays) plant.
Expression of a coding sequence by such a transformed plant (e.g.,
dicot or monocot) will result in the production of a pesticidal
polypeptide and confer increased insect resistance to the plant.
Some embodiments provide transgenic plants expressing pesticidal
polypeptides that find use in methods for impacting various insect
pests.
[0014] In another aspect, pesticidal or insecticidal compositions
containing the variant Cry1B polypeptides of the embodiments are
provided and the composition can optionally comprise further
insecticidal peptides. The embodiments encompass the application of
such compositions to the environment of insect pests in order to
impact the insect pests.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1a-1g shows an amino acid sequence alignment, using the
ALIGNX.RTM. module of the Vector NTI.RTM. suite, of Cry1Bd (SEQ ID
NO: 1), IP1B-B1 (SEQ ID NO: 3), IP1B-B21 (SEQ ID NO: 5), IP1B-B22
(SEQ ID NO: 7), IP1B-B23 (SEQ ID NO: 9), IP1B-B24 (SEQ ID NO: 11),
IP1B-B25 (SEQ ID NO: 13), IP1B-B26 (SEQ ID NO: 15), IP1B-B27 (SEQ
ID NO: 17), IP1B-B28 (SEQ ID NO: 19), IP1B-B29 (SEQ ID NO: 21),
IP1B-B31 (SEQ ID NO: 23), IP1B-B32 (SEQ ID NO: 25), IP1B-B33 (SEQ
ID NO: 27), IP1B-B34 (SEQ ID NO: 29), IP1B-B40 (SEQ ID NO: 31),
IP1B-B41 (SEQ ID NO: 33), IP1B-B42 (SEQ ID NO: 35), IP1B-B43 (SEQ
ID NO: 37), IP1B-B44 (SEQ ID NO: 39), IP1B-B45 (SEQ ID NO: 41),
IP1B-B46 (SEQ ID NO: 43), IP1B-B47 (SEQ ID NO: 45), MP258 (SEQ ID
NO: 47), and GS060 (SEQ ID NO: 49). The amino acid sequence
diversity between the Cry1B polypeptides is highlighted.
[0016] FIG. 2a-2e shows the amino acid sequence of MP258 with the
leader region (*), Domain I (#), Domain II (&), and Domain III
(!) indicated below the sequence.
[0017] FIG. 3 shows an amino acid sequence alignment, using the
ALIGNX.RTM. module of the Vector NTI.RTM. suite, of the Cry1Be type
Domain I of Cry1Be (amino acids 35-276 of SEQ ID NO: 58) and the
Cry1Be type Domain I of MP258 (amino acids 36-276 of SEQ ID NO:
47). The amino acid sequence diversity between Domains I of the
Cry1B polypeptides is highlighted.
[0018] FIG. 4 shows an amino acid sequence alignment, using the
ALIGNX.RTM. module of the Vector NTI.RTM. suite, of Domain III of
Cry1Ah (SEQ ID NO: 61), Cry1Bd, Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ
ID NO: 54), and MP258 (SEQ ID NO: 47). The amino acid sequence
diversity between Domain III the Cry1B polypeptides is
highlighted.
[0019] FIG. 5a-5c shows an amino acid sequence alignment, using the
ALIGNX.RTM. module of the Vector NTI.RTM. suite, of Domain I and
Domain II of MP258 (SEQ ID NO: 47), Cry1Be (SEQ ID NO: 58), Cry1Bi
(SEQ ID NO: 54), Cry1Bg (SEQ ID NO: 60), Cry1Bf (SEQ ID NO: 59),
Cry1Ba (SEQ ID NO: 55), Cry1Bh (SEQ ID NO: 52), Cry1Bd (SEQ ID NO:
1), Cry1Bb (SEQ ID NO: 56), and Cry1Bc (SEQ ID NO: 57). The amino
acid sequence diversity between Domain I and Domain II of the Cry1B
polypeptides is highlighted.
DETAILED DESCRIPTION
[0020] The embodiments of the disclosure are drawn to compositions
and methods for impacting insect pests, particularly plant pests.
More specifically, the isolated nucleic acid of the embodiments,
and fragments and variants thereof, comprise nucleotide sequences
that encode pesticidal polypeptides (e.g., proteins). The disclosed
pesticidal proteins are biologically active (e.g., pesticidal)
against insect pests such as, but not limited to, insect pests of
the order Lepidoptera and/or Coleoptera.
[0021] The compositions of the embodiments comprise isolated
nucleic acids, and fragments and variants thereof, which encode
pesticidal polypeptides, expression cassettes comprising nucleotide
sequences of the embodiments, isolated pesticidal proteins, and
pesticidal compositions. Some embodiments provide modified
pesticidal polypeptides having improved insecticidal activity
against Lepidopterans relative to the pesticidal activity of the
corresponding wild-type protein. The embodiments further provide
plants and microorganisms transformed with these novel nucleic
acids, and methods involving the use of such nucleic acids,
pesticidal compositions, transformed organisms, and products
thereof in impacting insect pests.
[0022] The nucleic acids and nucleotide sequences of the
embodiments may be used to transform any organism to produce the
encoded pesticidal proteins. Methods are provided that involve the
use of such transformed organisms to impact or control plant pests.
The nucleic acids and nucleotide sequences of the embodiments may
also be used to transform organelles such as chloroplasts (McBride
et al. (1995) Biotechnology 13: 362-365; and Kota et al. (1999)
Proc. Natl. Acad. Sci. USA 96: 1840-1845).
[0023] The embodiments further relate to the identification of
fragments and variants of the naturally-occurring coding sequence
that encode biologically active pesticidal proteins. The nucleotide
sequences of the embodiments find direct use in methods for
impacting pests, particularly insect pests such as pests of the
order Lepidoptera. Accordingly, the embodiments provide new
approaches for impacting insect pests that do not depend on the use
of traditional, synthetic chemical insecticides. The embodiments
involve the discovery of naturally-occurring, biodegradable
pesticides and the genes that encode them.
[0024] The embodiments further provide fragments and variants of
the naturally occurring coding sequence that also encode
biologically active (e.g., pesticidal) polypeptides. The nucleic
acids of the embodiments encompass nucleic acid or nucleotide
sequences that have been optimized for expression by the cells of a
particular organism, for example nucleic acid sequences that have
been back-translated (i.e., reverse translated) using
plant-preferred codons based on the amino acid sequence of a
polypeptide having enhanced pesticidal activity. The embodiments
further provide mutations which confer improved or altered
properties on the polypeptides of the embodiments. See, e.g. U.S.
Pat. No. 7,462,760.
[0025] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the embodiments.
[0026] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The above-defined terms are more
fully defined by reference to the specification as a whole.
[0027] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues (e.g., peptide nucleic acids) having the essential
nature of natural nucleotides in that they hybridize to
single-stranded nucleic acids in a manner similar to that of
naturally occurring nucleotides.
[0028] As used herein, the terms "encoding" or "encoded" when used
in the context of a specified nucleic acid mean that the nucleic
acid comprises the requisite information to direct translation of
the nucleotide sequence into a specified protein. The information
by which a protein is encoded is specified by the use of codons. A
nucleic acid encoding a protein may comprise non-translated
sequences (e.g., introns) within translated regions of the nucleic
acid or may lack such intervening non-translated sequences (e.g.,
as in cDNA).
[0029] As used herein, "full-length sequence" in reference to a
specified polynucleotide or its encoded protein means having the
entire nucleic acid sequence or the entire amino acid sequence of a
native (non-synthetic), endogenous sequence. A full-length
polynucleotide encodes the full-length, catalytically active form
of the specified protein.
[0030] As used herein, the term "antisense" used in the context of
orientation of a nucleotide sequence refers to a duplex
polynucleotide sequence that is operably linked to a promoter in an
orientation where the antisense strand is transcribed. The
antisense strand is sufficiently complementary to an endogenous
transcription product such that translation of the endogenous
transcription product is often inhibited. Thus, where the term
"antisense" is used in the context of a particular nucleotide
sequence, the term refers to the complementary strand of the
reference transcription product.
[0031] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0032] The terms "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogues of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0033] Polypeptides of the embodiments can be produced either from
a nucleic acid disclosed herein, or by the use of standard
molecular biology techniques. For example, a protein of the
embodiments can be produced by expression of a recombinant nucleic
acid of the embodiments in an appropriate host cell, or
alternatively by a combination of ex vivo procedures.
[0034] As used herein, the terms "isolated" and "purified" are used
interchangeably to refer to nucleic acids or polypeptides or
biologically active portions thereof that are substantially or
essentially free from components that normally accompany or
interact with the nucleic acid or polypeptide as found in its
naturally occurring environment. Thus, an isolated or purified
nucleic acid or polypeptide is substantially free of other cellular
material or culture medium when produced by recombinant techniques,
or substantially free of chemical precursors or other chemicals
when chemically synthesized.
[0035] An "isolated" nucleic acid is generally free of sequences
(such as, for example, protein-encoding sequences) that naturally
flank the nucleic acid (i.e., sequences located at the 5' and 3'
ends of the nucleic acid) in the genomic DNA of the organism from
which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acids can contain less than about
5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide
sequences that naturally flank the nucleic acids in genomic DNA of
the cell from which the nucleic acid is derived.
[0036] As used herein, the term "isolated" or "purified" as it is
used to refer to a polypeptide of the embodiments means that the
isolated protein is substantially free of cellular material and
includes preparations of protein having less than about 30%, 20%,
10%, or 5% (by dry weight) of contaminating protein. When the
protein of the embodiments or biologically active portion thereof
is recombinantly produced, culture medium represents less than
about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors
or non-protein-of-interest chemicals.
[0037] A "recombinant" nucleic acid molecule (or DNA) is used
herein to refer to a nucleic acid sequence (or DNA) that is in a
recombinant bacterial or plant host cell. In some embodiments, an
"isolated" or "recombinant" nucleic acid is free of sequences
(preferably protein encoding sequences) that naturally flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the
nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For purposes of the disclosure, "isolated"
or "recombinant" when used to refer to nucleic acid molecules
excludes isolated chromosomes.
[0038] As used herein a "non-genomic nucleic acid sequence" or
"non-genomic nucleic acid molecule" refers to a nucleic acid
molecule that has one or more change in the nucleic acid sequence
compared to a native or genomic nucleic acid sequence. In some
embodiments the change to a native or genomic nucleic acid molecule
includes but is not limited to: changes in the nucleic acid
sequence due to the degeneracy of the genetic code; codon
optimization of the nucleic acid sequence for expression in plants;
changes in the nucleic acid sequence to introduce at least one
amino acid substitution, insertion, deletion and/or addition
compared to the native or genomic sequence; removal of one or more
intron associated with the genomic nucleic acid sequence; insertion
of one or more heterologous introns; deletion of one or more
upstream or downstream regulatory regions associated with the
genomic nucleic acid sequence; insertion of one or more
heterologous upstream or downstream regulatory regions; deletion of
the 5' and/or 3' untranslated region associated with the genomic
nucleic acid sequence; insertion of a heterologous 5' and/or 3'
untranslated region; and modification of a polyadenylation site. In
some embodiments the non-genomic nucleic acid molecule is a cDNA.
In some embodiments the non-genomic nucleic acid molecule is a
synthetic nucleic acid sequence.
[0039] Throughout the specification the word "comprising," or
variations such as "comprises" or "comprising," will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0040] As used herein, the term "impacting insect pests" refers to
effecting changes in insect feeding, growth, and/or behavior at any
stage of development, including but not limited to: killing the
insect; retarding growth; preventing reproductive capability;
antifeedant activity; and the like.
[0041] As used herein, the terms "pesticidal activity" and
"insecticidal activity" are used synonymously to refer to activity
of an organism or a substance (such as, for example, a protein)
that can be measured by, but is not limited to, pest mortality,
pest weight loss, pest repellency, and other behavioral and
physical changes of a pest after feeding and exposure for an
appropriate length of time. Thus, an organism or substance having
pesticidal activity adversely impacts at least one measurable
parameter of pest fitness. For example, "pesticidal proteins" are
proteins that display pesticidal activity by themselves or in
combination with other proteins.
[0042] As used herein, the term "pesticidally effective amount"
means a quantity of a substance or organism that has pesticidal
activity when present in the environment of a pest. For each
substance or organism, the pesticidally effective amount is
determined empirically for each pest affected in a specific
environment. Similarly, an "insecticidally effective amount" may be
used to refer to a "pesticidally effective amount" when the pest is
an insect pest.
[0043] As used herein, the term "recombinantly engineered" or
"engineered" means the utilization of recombinant DNA technology to
introduce (e.g., engineer) a change in the protein structure based
on an understanding of the protein's mechanism of action and a
consideration of the amino acids being introduced, deleted, or
substituted.
[0044] As used herein, the term "mutant nucleotide sequence" or
"mutation" or "mutagenized nucleotide sequence" means a nucleotide
sequence that has been mutagenized or altered to contain one or
more nucleotide residues (e.g., base pair) that is not present in
the corresponding wild-type sequence. Such mutagenesis or
alteration consists of one or more additions, deletions, or
substitutions or replacements of nucleic acid residues. When
mutations are made by adding, removing, or replacing an amino acid
of a proteolytic site, such addition, removal, or replacement may
be within or adjacent to the proteolytic site motif, so long as the
object of the mutation is accomplished (i.e., so long as
proteolysis at the site is changed).
[0045] A mutant nucleotide sequence can encode a mutant
insecticidal toxin showing improved or decreased insecticidal
activity, or an amino acid sequence which confers improved or
decreased insecticidal activity on a polypeptide containing it. As
used herein, the term "mutant" or "mutation" in the context of a
protein a polypeptide or amino acid sequence refers to a sequence
which has been mutagenized or altered to contain one or more amino
acid residues that are not present in the corresponding wild-type
sequence. Such mutagenesis or alteration consists of one or more
additions, deletions, or substitutions or replacements of amino
acid residues. A mutant polypeptide shows improved or decreased
insecticidal activity, or represents an amino acid sequence which
confers improved insecticidal activity on a polypeptide containing
it. Thus, the term "mutant" or "mutation" refers to either or both
of the mutant nucleotide sequence and the encoded amino acids.
Mutants may be used alone or in any compatible combination with
other mutants of the embodiments or with other mutants. A "mutant
polypeptide" may conversely show a decrease in insecticidal
activity. Where more than one mutation is added to a particular
nucleic acid or protein, the mutations may be added at the same
time or sequentially; if sequentially, mutations may be added in
any suitable order.
[0046] As used herein, the term "improved insecticidal activity" or
"improved pesticidal activity" refers to an insecticidal
polypeptide of the embodiments that has enhanced insecticidal
activity relative to the activity of its corresponding wild-type
protein, and/or an insecticidal polypeptide that is effective
against a broader range of insects, and/or an insecticidal
polypeptide having specificity for an insect that is not
susceptible to the toxicity of the wild-type protein. A finding of
improved or enhanced pesticidal activity requires a demonstration
of an increase of pesticidal activity of at least 10%, against the
insect target, or at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
70%, 100%, 150%, 200%, or 300% or greater increase of pesticidal
activity relative to the pesticidal activity of the wild-type
insecticidal polypeptide determined against the same insect.
[0047] For example, an improved pesticidal or insecticidal activity
is provided where a wider or narrower range of insects is impacted
by the polypeptide relative to the range of insects that is
affected by a wild-type Bt toxin. A wider range of impact may be
desirable where versatility is desired, while a narrower range of
impact may be desirable where, for example, beneficial insects
might otherwise be impacted by use or presence of the toxin. While
the embodiments are not bound by any particular mechanism of
action, an improved pesticidal activity may also be provided by
changes in one or more characteristics of a polypeptide; for
example, the stability or longevity of a polypeptide in an insect
gut may be increased relative to the stability or longevity of a
corresponding wild-type protein.
[0048] The term "toxin" as used herein refers to a polypeptide
showing pesticidal activity or insecticidal activity or improved
pesticidal activity or improved insecticidal activity. "Bt" or
"Bacillus thuringiensis" toxin is intended to include the broader
class of Cry toxins found in various strains of Bt, which includes
such toxins as, for example, Cry1s, Cry2s, or Cry3s.
[0049] The terms "proteolytic site" or "cleavage site" refer to an
amino acid sequence which confers sensitivity to a class of
proteases or a particular protease such that a polypeptide
containing the amino acid sequence is digested by the class of
proteases or particular protease. A proteolytic site is said to be
"sensitive" to the protease(s) that recognize that site. It is
appreciated in the art that the efficiency of digestion will vary,
and that a decrease in efficiency of digestion can lead to an
increase in stability or longevity of the polypeptide in an insect
gut. Thus, a proteolytic site may confer sensitivity to more than
one protease or class of proteases, but the efficiency of digestion
at that site by various proteases may vary. Proteolytic sites
include, for example, trypsin sites, chymotrypsin sites, and
elastase sites.
[0050] Research has shown that the insect gut proteases of
Lepidopterans include trypsins, chymotrypsins, and elastases. See,
e.g., Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16:
201-212; and Hedegus et al. (2003) Arch. Insect Biochem. Physiol.
53: 30-47. For example, about 18 different trypsins have been found
in the midgut of Helicoverpa armigera larvae (see Gatehouse et al.
(1997) Insect Biochem. Mol. Biol. 27: 929-944). The preferred
proteolytic substrate sites of these proteases have been
investigated. See, e.g., Peterson et al. (1995) Insect Biochem.
Mol. Biol. 25: 765-774.
[0051] Efforts have been made to understand the mechanism of action
of Bt toxins and to engineer toxins with improved properties. It
has been shown that insect gut proteases can affect the impact of
Bt Cry proteins on the insect. Some proteases activate the Cry
proteins by processing them from a "protoxin" form into a toxic
form, or "toxin." See, Oppert (1999) Arch. Insect Biochem. Phys.
42: 1-12; and Carroll et al. (1997) J. Invertebrate Pathology 70:
41-49. This activation of the toxin can include the removal of the
N- and C-terminal peptides from the protein and can also include
internal cleavage of the protein. Other proteases can degrade the
Cry proteins. See Oppert, ibid.
[0052] A comparison of the amino acid sequences of Cry toxins of
different specificities reveals five highly-conserved sequence
blocks. Structurally, the toxins comprise three distinct Domains
which are, from the N- to C-terminus: a cluster of seven
alpha-helices implicated in pore formation (referred to as "Domain
I"), three anti-parallel beta sheets implicated in cell binding
(referred to as "Domain 2"), and a beta sandwich (referred to as
"Domain 3"). The location and properties of these Domains are known
to those of skill in the art. See, for example, Li et al. (1991)
Nature, 305:815-821 and Morse et al. (2001) Structure, 9:409-417.
When reference is made to a particular domain, such as Domain I, it
is understood that the exact endpoints of the domain with regard to
a particular sequence are not critical so long as the sequence or
portion thereof includes sequence that provides at least some
function attributed to the particular domain. Thus, for example,
when referring to "Domain I," it is intended that a particular
sequence includes a cluster of seven alpha-helices, but the exact
endpoints of the sequence used or referred to with regard to that
cluster are not critical. One of skill in the art is familiar with
the determination of such endpoints and the evaluation of such
functions.
[0053] In an effort to improve Cry2B toxins, an effort was
undertaken to identify the nucleotide sequences encoding the
crystal proteins from the selected strains, which had improved
activity compared to the native toxin. Depending upon the
characteristics of a given preparation, it was recognized that the
demonstration of pesticidal activity sometimes required trypsin
pretreatment to activate the pesticidal proteins. Thus, it is
understood that some pesticidal proteins require protease digestion
(e.g., by trypsin, chymotrypsin, and the like) for activation,
while other proteins are biologically active (e.g., pesticidal) in
the absence of activation.
[0054] Such molecules may be altered by means described, for
example, U.S. Pat. No. 7,462,760. In addition, nucleic acid
sequences may be engineered to encode polypeptides that contain
additional mutations that confer improved or altered pesticidal
activity relative to the pesticidal activity of the naturally
occurring polypeptide. The nucleotide sequences of such engineered
nucleic acids comprise mutations not found in the wild type
sequences.
[0055] The mutant polypeptides of the embodiments are generally
prepared by a process that involves the steps of: obtaining a
nucleic acid sequence encoding a Cry family polypeptide; analyzing
the structure of the polypeptide to identify particular "target"
sites for mutagenesis of the underlying gene sequence based on a
consideration of the proposed function of the target domain in the
mode of action of the toxin; introducing one or more mutations into
the nucleic acid sequence to produce a desired change in one or
more amino acid residues of the encoded polypeptide sequence; and
assaying the polypeptide produced for pesticidal activity.
[0056] Many of the Bt insecticidal toxins are related to various
degrees by similarities in their amino acid sequences and tertiary
structure and means for obtaining the crystal structures of Bt
toxins are well known. Exemplary high-resolution crystal structure
solution of both the Cry3A and Cry3B polypeptides are available in
the literature. The solved structure of Cry3A (Li et al. (1991)
Nature 353:815-821) provides insight into the relationship between
structure and function of the toxin. A combined consideration of
the published structural analyses of Bt toxins and the reported
function associated with particular structures, motifs, and the
like indicates that specific regions of the toxin are correlated
with particular functions and discrete steps of the mode of action
of the protein. For example, many toxins isolated from Bt are
generally described as comprising three domains: a seven-helix
bundle that is involved in pore formation, a three-sheet domain
that has been implicated in receptor binding, and a beta-sandwich
motif (Li et al. (1991) Nature 305: 815-821).
[0057] As reported in U.S. Pat. Nos. 7,105,332, and 7,462,760, the
toxicity of Cry proteins can be improved by targeting the region
located between alpha helices 3 and 4 of Domain I of the toxin.
This theory was premised on a body of knowledge concerning
insecticidal toxins, including: 1) that alpha helices 4 and 5 of
Domain I of Cry3A toxins had been reported to insert into the lipid
bilayer of cells lining the midgut of susceptible insects (Gazit et
al. (1998) Proc. Natl. Acad. Sci. USA 95: 12289-12294); 2) the
inventors' knowledge of the location of trypsin and chymotrypsin
cleavage sites within the amino acid sequence of the wild-type
protein; 3) the observation that the wild-type protein was more
active against certain insects following in vitro activation by
trypsin or chymotrypsin treatment; and 4) reports that digestion of
toxins from the 3' end resulted in decreased toxicity to
insects.
[0058] A series of mutations may be created and placed in a variety
of background sequences to create novel polypeptides having
enhanced or altered pesticidal activity. See, e.g., U.S. Pat. No.
7,462,760. These mutants include, but are not limited to: the
addition of at least one more protease-sensitive site (e.g.,
trypsin cleavage site) in the region located between helices 3 and
4 of Domain I; the replacement of an original protease-sensitive
site in the wild-type sequence with a different protease-sensitive
site; the addition of multiple protease-sensitive sites in a
particular location; the addition of amino acid residues near
protease-sensitive site(s) to alter folding of the polypeptide and
thus enhance digestion of the polypeptide at the protease-sensitive
site(s); and adding mutations to protect the polypeptide from
degradative digestion that reduces toxicity (e.g., making a series
of mutations wherein the wild-type amino acid is replaced by valine
to protect the polypeptide from digestion). Mutations may be used
singly or in any combination to provide polypeptides of the
embodiments.
[0059] Homologous sequences were identified by similarity search on
the non-redundant database (nr) of National Center for
Bioinformatics Information (NCBI) using BLAST and PSI-BLAST. The
homologous proteins were made up of Cry toxins primarily from
Bacillus thuringiensis.
[0060] A mutation which is an additional or alternative
protease-sensitive site may be sensitive to several classes of
proteases such as serine proteases, which include trypsin and
chymotrypsin, or enzymes such as elastase. Thus, a mutation which
is an additional or alternative protease-sensitive site may be
designed so that the site is readily recognized and/or cleaved by a
category of proteases, such as mammalian proteases or insect
proteases. A protease-sensitive site may also be designed to be
cleaved by a particular class of enzymes or a particular enzyme
known to be produced in an organism, such as, for example, a
chymotrypsin produced by the corn earworm Heliothis zea (Lenz et
al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212). Mutations
may also confer resistance to proteolytic digestion, for example,
to digestion by chymotrypsin at the C-terminus of the peptide.
[0061] The presence of an additional and/or alternative
protease-sensitive site in the amino acid sequence of the encoded
polypeptide can improve the pesticidal activity and/or specificity
of the polypeptide encoded by the nucleic acids of the embodiments.
Accordingly, the nucleotide sequences of the embodiments can be
recombinantly engineered or manipulated to produce polypeptides
having improved or altered insecticidal activity and/or specificity
compared to that of an unmodified wild-type toxin. In addition, the
mutations disclosed herein may be placed in or used in conjunction
with other nucleotide sequences to provide improved properties. For
example, a protease-sensitive site that is readily cleaved by
insect chymotrypsin, e.g., a chymotrypsin found in the bertha
armyworm or the corn earworm (Hegedus et al. (2003) Arch. Insect
Biochem. Physiol. 53: 30-47; and Lenz et al. (1991) Arch. Insect
Biochem. Physiol. 16: 201-212), may be placed in a Cry background
sequence to provide improved toxicity to that sequence. In this
manner, the embodiments provide toxic polypeptides with improved
properties.
[0062] For example, a mutagenized Cry nucleotide sequence can
comprise additional mutants that comprise additional codons that
introduce a second trypsin-sensitive amino acid sequence (in
addition to the naturally occurring trypsin site) into the encoded
polypeptide. An alternative addition mutant of the embodiments
comprises additional codons designed to introduce at least one
additional different protease-sensitive site into the polypeptide,
for example, a chymotrypsin-sensitive site located immediately 5'
or 3' of the naturally occurring trypsin site. Alternatively,
substitution mutants may be created in which at least one codon of
the nucleic acid that encodes the naturally occurring
protease-sensitive site is destroyed and alternative codons are
introduced into the nucleic acid sequence in order to provide a
different (e.g., substitute) protease-sensitive site. A replacement
mutant may also be added to a Cry sequence in which the
naturally-occurring trypsin cleavage site present in the encoded
polypeptide is destroyed and a chymotrypsin or elastase cleavage
site is introduced in its place.
[0063] It is recognized that any nucleotide sequence encoding the
amino acid sequences that are proteolytic sites or putative
proteolytic sites (for example, sequences such as RR, or LKM) can
be used and that the exact identity of the codons used to introduce
any of these cleavage sites into a variant polypeptide may vary
depending on the use, i.e., expression in a particular plant
species. It is also recognized that any of the disclosed mutations
can be introduced into any polynucleotide sequence of the
embodiments that comprises the codons for amino acid residues that
provide the native trypsin cleavage site that is targeted for
modification. Accordingly, variants of either full-length toxins or
fragments thereof can be modified to contain additional or
alternative cleavage sites, and these embodiments are intended to
be encompassed by the scope of the embodiments disclosed
herein.
[0064] It will be appreciated by those of skill in the art that any
useful mutation may be added to the sequences of the embodiments so
long as the encoded polypeptides retain pesticidal activity. Thus,
sequences may also be mutated so that the encoded polypeptides are
resistant to proteolytic digestion by chymotrypsin. More than one
recognition site can be added in a particular location in any
combination, and multiple recognition sites can be added to or
removed from the toxin. Thus, additional mutations can comprise
three, four, or more recognition sites. It is to be recognized that
multiple mutations can be engineered in any suitable polynucleotide
sequence; accordingly, either full-length sequences or fragments
thereof can be modified to contain additional or alternative
cleavage sites as well as to be resistant to proteolytic digestion.
In this manner, the embodiments provide Cry toxins containing
mutations that improve pesticidal activity as well as improved
compositions and methods for impacting pests using other Bt
toxins.
[0065] Mutations may protect the polypeptide from protease
degradation, for example by removing putative proteolytic sites
such as putative serine protease sites and elastase recognition
sites from different areas. Some or all of such putative sites may
be removed or altered so that proteolysis at the location of the
original site is decreased. Changes in proteolysis may be assessed
by comparing a mutant polypeptide with wild-type toxins or by
comparing mutant toxins which differ in their amino acid sequence.
Putative proteolytic sites and proteolytic sites include, but are
not limited to, the following sequences: RR, a trypsin cleavage
site; LKM, a chymotrypsin site; and a trypsin site. These sites may
be altered by the addition or deletion of any number and kind of
amino acid residues, so long as the pesticidal activity of the
polypeptide is increased. Thus, polypeptides encoded by nucleotide
sequences comprising mutations will comprise at least one amino
acid change or addition relative to the native or background
sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38,
40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 or
more amino acid changes or additions. Pesticidal activity of a
polypeptide may also be improved by truncation of the native or
full-length sequence, as is known in the art.
[0066] Compositions of the embodiments include nucleic acids, and
fragments and variants thereof that encode pesticidal polypeptides.
In particular, the embodiments provide for isolated nucleic acid
molecules comprising nucleotide sequences encoding the amino acid
sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID
NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 and SEQ ID NO:
45, or the nucleotide sequences encoding said amino acid sequence,
for example the nucleotide sequence set forth in SEQ ID NO: 4, SEQ
ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:
14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ
ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:
32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ
ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46, and fragments and
variants thereof.
[0067] In particular, the embodiments provide for isolated nucleic
acid molecules encoding the amino acid sequence shown in SEQ ID NO:
4 or SEQ ID NO: 8, or the nucleotide sequences encoding said amino
acid sequence, for example the nucleotide sequence set forth in SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 46, and
fragments and variants thereof.
[0068] Also of interest are optimized nucleotide sequences encoding
the pesticidal proteins of the embodiments. As used herein, the
phrase "optimized nucleotide sequences" refers to nucleic acids
that are optimized for expression in a particular organism, for
example a plant. Optimized nucleotide sequences may be prepared for
any organism of interest using methods known in the art. See, for
example, U.S. Pat. No. 7,462,760, which describes an optimized
nucleotide sequence encoding a disclosed pesticidal protein. In
this example, the nucleotide sequence was prepared by
reverse-translating the amino acid sequence of the protein and
changing the nucleotide sequence so as to comprise maize-preferred
codons while still encoding the same amino acid sequence. This
procedure is described in more detail by Murray et al. (1989)
Nucleic Acids Res. 17:477-498. Optimized nucleotide sequences find
use in increasing expression of a pesticidal protein in a plant,
for example monocot plants of the Gramineae (Poaceae) family such
as, for example, a maize or corn plant.
[0069] In some embodiments polypeptides are provided comprising an
amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID
NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID
NO: 43 or SEQ ID NO: 45 and fragments and variants thereof.
[0070] In some embodiments polypeptides are provided comprising an
amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID
NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15,
SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43 or SEQ ID NO: 45 and fragments and variants
thereof.
[0071] In some embodiments polypeptides are provided comprising an
amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 25, SEQ
ID NO: 27 or SEQ ID NO: 29, and fragments and variants thereof.
[0072] In some embodiments variant Cry1B polypeptides having an
amino acid substitution compared to the corresponding reference
Cry1B polypeptide are provides that have increased insecticidal
activity against corn earworm and/or fall armyworm compared to the
"corresponding reference Cry1B polypeptide". By "corresponding
reference Cry1B polypeptide" is meant a wild type or native Cry1B
polypeptide or variant Cry1B polypeptide of the present
embodiments, which can serve as the amino acid sequence that is
mutagenized to create variant Cry1B polypeptide. In some
embodiments the corresponding reference Cry1B polypeptide comprises
a Cry1Be type Domain I and a Cry1Ah type Domain III. By "Cry1Be
type Domain I" is meant an amino acid sequence having at least 90%,
at least 91%, at least 92% at least 93% at least 94%, at least 95%
at least 96%, at least 97%, at least 98%, at least 99% or greater
sequence identity to amino acids 36-276 of SEQ ID NO: 58 (Cry1Be)
or amino acids 35-276 of SEQ ID NO: 47. An amino acid sequence
alignment of Domain I of Cry1Be (SEQ ID NO: 58) and MP258 (SEQ ID
NO: 47) is shown in FIG. 3. Similarly, other native Cry1B
polypeptides can be aligned with Cry1Be (SEQ ID NO: 58) and MP258
(SEQ ID NO: 47) to identify other Cry1Be type Domain I regions. By
"Cry1Ah type Domain III" is meant an amino acid sequence having at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%,
at least 85%, at least 86%, at least 87%, at least 88%, at least
89%, at least 90%, at least 91%, at least 92% at least 93% at least
94%, at least 95% at least 96%, at least 97%, at least 98%, at
least 99% or greater sequence identity to amino acids 483-643 of
SEQ ID NO: 61 (Cry1Ah) or 494-655 of SEQ ID NO: 47. An amino acid
sequence alignment of Domain III of Cry1Ah (SEQ ID NO: 61), Cry1Bd
(SEQ ID NO: 1), Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ ID NO: 54), and
MP258 (SEQ ID NO: 47) is shown in FIG. 4. Similarly, other native
Cry1B polypeptides can be aligned with Cry1Ah (SEQ ID NO: 61),
Cry1Bd, Cry1Bh (SEQ ID NO: 52), Cry1Bi (SEQ ID NO: 54), and/or
MP258 (SEQ ID NO: 47) to identify other Cry1Ah type Domain III
regions. In some embodiments the corresponding reference Cry1B
polypeptide comprises a Cry1Ba type Domain I and Domain II. By
"Cry1Ba type Domain I and Domain II" is meant an amino acid
sequence having at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92% at least 93% at least 94%, at least 95% at least 96%, at
least 97%, at least 98%, at least 99% or greater sequence identity
to amino acids 30-489 of SEQ ID NO: 55 (Cry1Ba). An amino acid
sequence alignment of Domain I and Domain II of MP258 (SEQ ID NO:
47), Cry1Be (SEQ ID NO: 58), Cry1Bi (SEQ ID NO: 54), Cry1Bg (SEQ ID
NO: 60), Cry1Bf (SEQ ID NO: 59), Cry1Ba (SEQ ID NO: 55), Cry1Bh
(SEQ ID NO: 52), Cry1Bd (SEQ ID NO: 1), Cry1Bb (SEQ ID NO: 56), and
Cry1Bc (SEQ ID NO: 57) is shown in FIG. 5. Similarly, other native
Cry1B polypeptides can be aligned with Cry1Ba (SEQ ID NO: 55) and
MP258 (SEQ ID NO: 47) to identify other Cry1Ba type Domain I and
Domain II regions.
[0073] In some embodiments the corresponding reference Cry1B
polypeptide comprises a Cry1Be type Domain I and Domain II. By
"Cry1Be type Domain I and Domain II" is meant an amino acid
sequence having at least 70%, at least 71%, at least 72%, at least
73%, at least 74%, at least 75%, at least 76%, at least 77%, at
least 78%, at least 79%, at least 80%, at least 81%, at least 82%,
at least 83%, at least 84%, at least 85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at
least 92% at least 93% at least 94%, at least 95% at least 96%, at
least 97%, at least 98%, at least 99% or greater sequence identity
to amino acids 35-494 of SEQ ID NO: 58 (Cry1Be) or amino acids
35-493 of SEQ ID NO: 47. An amino acid sequence alignment of Domain
I and Domain II of MP258 (SEQ ID NO: 47), Cry1Be (SEQ ID NO: 58),
Cry1Bi (SEQ ID NO: 54), Cry1Bg (SEQ ID NO: 60), Cry1Bf (SEQ ID NO:
59), Cry1Ba (SEQ ID NO: 55), Cry1Bh (SEQ ID NO: 52), Cry1Bd (SEQ ID
NO: 1), Cry1Bb (SEQ ID NO: 56), and Cry1Bc (SEQ ID NO: 57) is shown
in FIG. 5. Similarly, other native Cry1B polypeptides can be
aligned with Cry1Be (SEQ ID NO: 58) and MP258 (SEQ ID NO: 47) to
identify other Cry1Be type Domain I and Domain II regions. By
"improved activity" or "increased activity" is intended an increase
of at least about 10%, at least about 15%, at least about 20%, at
least about 25%, at least about 30%, at least about 35%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 100%,
at least about 110%, at least about 120%, at least about 130%, at
least about 140%, at least about 150%, at least about 160%, at
least about 170%, at least about 180%, at least about 190%, at
least about 200%, at least about 210% at least about 220%, at least
about 230%, at least about 240%, at least about 250%, at least
about 260%, at least about 270%, at least about 280%, at least
about 290%, at least about 300%, at least about 310%, at least
about 320%, at least about 330%, at least about 340%, at least
about 350%, at least about 360%, at least about 370%, at least
about 380%, at least about 390%, at least about 400%, at least
about 410%, at least about 420%, at least about 430%, at least
about 440%, at least about 450%, at least about 460%, at least
about 470%, at least about 480%, at least about 490%, at least
about 500%, at least about 510%, at least about 520%, at least
about 530%, at least about 540%, at least about 550%, at least
about 560%, at least about 570%, at least about 580%, at least
about 590%, at least about 600%, at least about 650%, at least
about 700%, at least about 750%, at least about 800%, at least
about 850%, at least about 900%, at least about 950%, at least
about 1000% or higher or at least about 1.1-fold, at least about
1.2-fold, at least about 1.3-fold, at least about 1.4-fold or at
least about 1.5-fold, at least about 1.6-fold, at least about
1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at
least about 2-fold, at least about 2.1-fold, at least about
2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at
least about 2.5-fold, at least about 2.6-fold, at least about
2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at
least about 3-fold, at least about 3.1-fold, at least about
3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at
least about 3.5-fold, at least about 3.6-fold, at least about
3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at
least about 4-fold, at least about 4.1-fold, at least about
4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at
least about 4.5-fold, at least about 4.6-fold, at least about
4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at
least about 5-fold, at least about 5.1-fold, at least about
5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at
least about 5.5-fold, at least about 5.6-fold, at least about
5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at
least about 6-fold, at least about 6.1-fold, at least about
6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at
least about 6.5-fold, at least about 6.6-fold, at least about
6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at
least about 7-fold, at least about 7.1-fold, at least about
7.2-fold, at least about 7.3-fold, at least about 7.4-fold, at
least about 7.5-fold, at least about 7.6-fold, at least about
7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at
least about 8-fold, at least about 8.1-fold, at least about
8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at
least about 8.5-fold, at least about 8.6-fold, at least about
8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at
least about 9-fold, at least about 9.1-fold, at least about
9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at
least about 9.5-fold, at least about 9.6-fold, at least about
9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at
least about 10-fold or higher increase in the pesticidal activity
of the variant protein compared to the activity of the
corresponding reference Cry1B polypeptide.
[0074] In some embodiments, the improvement consists of a decrease
in the EC50 of at least about 10%, at least about 15%, at least
about 20%, at least about 25%, at least about 30%, at least about
35%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about 80%, at least about 90%, at least
about 100%, at least about 110%, at least about 120%, at least
about 130%, at least about 140%, at least about 150%, at least
about 160%, at least about 170%, at least about 180%, at least
about 190%, at least about 200%, at least about 210% at least about
220%, at least about 230%, at least about 240%, at least about
250%, at least about 260%, at least about 270%, at least about
280%, at least about 290%, at least about 300%, at least about
310%, at least about 320%, at least about 330%, at least about
340%, at least about 350%, at least about 360%, at least about
370%, at least about 380%, at least about 390%, at least about
400%, at least about 410%, at least about 420%, at least about
430%, at least about 440%, at least about 450%, at least about
460%, at least about 470%, at least about 480%, at least about
490%, at least about 500%, at least about 510%, at least about
520%, at least about 530%, at least about 540%, at least about
550%, at least about 560%, at least about 570%, at least about
580%, at least about 590%, at least about 600%, at least about
650%, at least about 700%, at least about 750%, at least about
800%, at least about 850%, at least about 900%, at least about
950%, at least about 1000% or higher or at least about 1.1-fold, at
least about 1.2-fold, at least about 1.3-fold, at least about
1.4-fold or at least about 1.5-fold, at least about 1.6-fold, at
least about 1.7-fold, at least about 1.8-fold, at least about
1.9-fold, at least about 2-fold, at least about 2.1-fold, at least
about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold,
at least about 2.5-fold, at least about 2.6-fold, at least about
2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at
least about 3-fold, at least about 3.1-fold, at least about
3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at
least about 3.5-fold, at least about 3.6-fold, at least about
3.7-fold, at least about 3.8-fold, at least about 3.9-fold, at
least about 4-fold, at least about 4.1-fold, at least about
4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at
least about 4.5-fold, at least about 4.6-fold, at least about
4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at
least about 5-fold, at least about 5.1-fold, at least about
5.2-fold, at least about 5.3-fold, at least about 5.4-fold, at
least about 5.5-fold, at least about 5.6-fold, at least about
5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at
least about 6-fold, at least about 6.1-fold, at least about
6.2-fold, at least about 6.3-fold, at least about 6.4-fold, at
least about 6.5-fold, at least about 6.6-fold, at least about
6.7-fold, at least about 6.8-fold, at least about 6.9-fold, at
least about 7-fold, at least about 7.1-fold, at least about
7.2-fold, at least about 7.3-fold, at least about 7.4-fold, at
least about 7.5-fold, at least about 7.6-fold, at least about
7.7-fold, at least about 7.8-fold, at least about 7.9-fold, at
least about 8-fold, at least about 8.1-fold, at least about
8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at
least about 8.5-fold, at least about 8.6-fold, at least about
8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at
least about 9-fold, at least about 9.1-fold, at least about
9.2-fold, at least about 9.3-fold, at least about 9.4-fold, at
least about 9.5-fold, at least about 9.6-fold, at least about
9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at
least about 10-fold or greater reduction in the EC50 of the variant
Cry1B polypeptide relative to the pesticidal activity of the
corresponding reference Cry1B polypeptide.
[0075] In some embodiments the EC50 of the variant Cry1B
polypeptide is <100 ppm, <90 ppm, <80 ppm, <70 ppm,
<60 ppm, <50 ppm, <45 ppm, <40 ppm, <35 ppm, <30
ppm, <25 ppm, <20 ppm, <19 ppm, <18 ppm, <17 ppm,
<16 ppm, <15 ppm, <14 ppm, <13 ppm, <12 ppm, <11
ppm, <10 ppm, <9 ppm, <8 ppm, <7 ppm, <6 ppm, <5
ppm, <4 ppm, <3 ppm, <2 ppm, <1 ppm, <0.9 ppm,
<0.8 ppm, <0.7 ppm, <0.6 ppm, <0.5 ppm, <0.4 ppm,
<0.3 ppm, <0.2 ppm or <0.1 ppm.
[0076] In some embodiments, the improvement consists of an increase
in the Mean FAE Index of at least about 10%, at least about 15%, at
least about 20%, at least about 25%, at least about 30%, at least
about 35%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 100%, at least about 110%, at least about 120%, at
least about 130%, at least about 140%, at least about 150%, at
least about 160%, at least about 170%, at least about 180%, at
least about 190%, at least about 200%, at least about 210% at least
about 220%, at least about 230%, at least about 240%, at least
about 250%, at least about 260%, at least about 270%, at least
about 280%, at least about 290%, at least about 300%, at least
about 310%, at least about 320%, at least about 330%, at least
about 340%, at least about 350%, at least about 360%, at least
about 370%, at least about 380%, at least about 390%, at least
about 400%, at least about 410%, at least about 420%, at least
about 430%, at least about 440%, at least about 450%, at least
about 460%, at least about 470%, at least about 480%, at least
about 490%, at least about 500%, at least about 510%, at least
about 520%, at least about 530%, at least about 540%, at least
about 550%, at least about 560%, at least about 570%, at least
about 580%, at least about 590%, at least about 600%, at least
about 650%, at least about 700%, at least about 750%, at least
about 800%, at least about 850%, at least about 900%, at least
about 950%, at least about 1000% or higher or at least about
1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at
least about 1.4-fold or at least about 1.5-fold, at least about
1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at
least about 1.9-fold, at least about 2-fold, at least about
2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at
least about 2.4-fold, at least about 2.5-fold, at least about
2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at
least about 2.9-fold, at least about 3-fold, at least about
3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at
least about 3.4-fold, at least about 3.5-fold, at least about
3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at
least about 3.9-fold, at least about 4-fold, at least about
4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at
least about 4.4-fold, at least about 4.5-fold, at least about
4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at
least about 4.9-fold, at least about 5-fold, at least about
5.1-fold, at least about 5.2-fold, at least about 5.3-fold, at
least about 5.4-fold, at least about 5.5-fold, at least about
5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at
least about 5.9-fold, at least about 6-fold, at least about
6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at
least about 6.4-fold, at least about 6.5-fold, at least about
6.6-fold, at least about 6.7-fold, at least about 6.8-fold, at
least about 6.9-fold, at least about 7-fold, at least about
7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at
least about 7.4-fold, at least about 7.5-fold, at least about
7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at
least about 7.9-fold, at least about 8-fold, at least about
8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at
least about 8.4-fold, at least about 8.5-fold, at least about
8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at
least about 8.9-fold, at least about 9-fold, at least about
9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at
least about 9.4-fold, at least about 9.5-fold, at least about
9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at
least about 9.9-fold, at least about 10-fold or higher increase in
the Mean FAE Index of the variant Cry1B polypeptide relative to the
pesticidal activity of the corresponding reference Cry1B
polypeptide.
[0077] "Mean FAE Index" (MFI) refers to the mean of multiple FAEGN
an arithmetic mean of FAEGN. As used herein, the "Mean Deviation
Score" refers to the arithmetic mean of multiple Deviation
Scores.
[0078] In some embodiments, the improvement consists of an increase
in the Mean Deviation Score of at least about 10%, at least about
15%, at least about 20%, at least about 25%, at least about 30%, at
least about 35%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 100%, at least about 110%, at least about 120%,
at least about 130%, at least about 140%, at least about 150%, at
least about 160%, at least about 170%, at least about 180%, at
least about 190%, at least about 200%, at least about 210% at least
about 220%, at least about 230%, at least about 240%, at least
about 250%, at least about 260%, at least about 270%, at least
about 280%, at least about 290%, at least about 300%, at least
about 310%, at least about 320%, at least about 330%, at least
about 340%, at least about 350%, at least about 360%, at least
about 370%, at least about 380%, at least about 390%, at least
about 400%, at least about 410%, at least about 420%, at least
about 430%, at least about 440%, at least about 450%, at least
about 460%, at least about 470%, at least about 480%, at least
about 490%, at least about 500%, at least about 510%, at least
about 520%, at least about 530%, at least about 540%, at least
about 550%, at least about 560%, at least about 570%, at least
about 580%, at least about 590%, at least about 600%, at least
about 650%, at least about 700%, at least about 750%, at least
about 800%, at least about 850%, at least about 900%, at least
about 950%, at least about 1000% or higher or at least about
1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at
least about 1.4-fold or at least about 1.5-fold, at least about
1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at
least about 1.9-fold, at least about 2-fold, at least about
2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at
least about 2.4-fold, at least about 2.5-fold, at least about
2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at
least about 2.9-fold, at least about 3-fold, at least about
3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at
least about 3.4-fold, at least about 3.5-fold, at least about
3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at
least about 3.9-fold, at least about 4-fold, at least about
4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at
least about 4.4-fold, at least about 4.5-fold, at least about
4.6-fold, at least about 4.7-fold, at least about 4.8-fold, at
least about 4.9-fold, at least about 5-fold, at least about
5.1-fold, at least about 5.2-fold, at least about 5.3-fold, at
least about 5.4-fold, at least about 5.5-fold, at least about
5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at
least about 5.9-fold, at least about 6-fold, at least about
6.1-fold, at least about 6.2-fold, at least about 6.3-fold, at
least about 6.4-fold, at least about 6.5-fold, at least about
6.6-fold, at least about 6.7-fold, at least about 6.8-fold, at
least about 6.9-fold, at least about 7-fold, at least about
7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at
least about 7.4-fold, at least about 7.5-fold, at least about
7.6-fold, at least about 7.7-fold, at least about 7.8-fold, at
least about 7.9-fold, at least about 8-fold, at least about
8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at
least about 8.4-fold, at least about 8.5-fold, at least about
8.6-fold, at least about 8.7-fold, at least about 8.8-fold, at
least about 8.9-fold, at least about 9-fold, at least about
9.1-fold, at least about 9.2-fold, at least about 9.3-fold, at
least about 9.4-fold, at least about 9.5-fold, at least about
9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at
least about 9.9-fold, at least about 10-fold or higher increase in
the Mean Deviation Score of the variant Cry1B polypeptide relative
to the pesticidal activity of the corresponding reference Cry1B
polypeptide.
[0079] In some embodiments the improved activity of the variant
Cry1B polypeptide is relative to the pesticidal activity of SEQ ID
NO: 1 (Cry1Bd), SEQ ID NO: 47 (MP258), SEQ ID NO: 52 (Cry1Bh), SEQ
ID NO: 54 (Cry1Bi), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ
ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:
17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43 or SEQ ID NO: 45.
[0080] In particular embodiments, pesticidal proteins of the
embodiments provide full-length insecticidal polypeptides,
fragments of full-length insecticidal polypeptides, and variant
polypeptides that are produced from mutagenized nucleic acids
designed to introduce particular amino acid sequences into
polypeptides of the embodiments. In particular embodiments, the
amino acid sequences that are introduced into the polypeptides
comprise a sequence that provides a cleavage site for an enzyme
such as a protease.
[0081] It is known in the art that the pesticidal activity of Bt
toxins is typically activated by cleavage of the peptide in the
insect gut by various proteases. Because peptides may not always be
cleaved with complete efficiency in the insect gut, fragments of a
full-length toxin may have enhanced pesticidal activity in
comparison to the full-length toxin itself. Thus, some of the
polypeptides of the embodiments include fragments of a full-length
insecticidal polypeptide, and some of the polypeptide fragments,
variants, and mutations will have enhanced pesticidal activity
relative to the activity of the naturally occurring insecticidal
polypeptide from which they are derived, particularly if the
naturally occurring insecticidal polypeptide is not activated in
vitro with a protease prior to screening for activity. Thus, the
present application encompasses truncated versions or fragments of
the sequences.
[0082] Mutations may be placed into any background sequence,
including such truncated polypeptides, so long as the polypeptide
retains pesticidal activity. One of skill in the art can readily
compare two or more proteins with regard to pesticidal activity
using assays known in the art or described elsewhere herein. It is
to be understood that the polypeptides of the embodiments can be
produced either by expression of a nucleic acid disclosed herein,
or by the use of standard molecular biology techniques.
[0083] It is recognized that the pesticidal proteins may be
oligomeric and will vary in molecular weight, number of residues,
component peptides, activity against particular pests, and other
characteristics. However, by the methods set forth herein, proteins
active against a variety of pests may be isolated and
characterized. The pesticidal proteins of the embodiments can be
used in combination with other Bt toxins or other insecticidal
proteins to increase insect target range. Furthermore, the use of
the pesticidal proteins of the embodiments in combination with
other Bt toxins or other insecticidal principles of a distinct
nature has particular utility for the prevention and/or management
of insect resistance. Other insecticidal agents include protease
inhibitors (both serine and cysteine types), .alpha.-amylase, and
peroxidase.
[0084] Fragments and variants of the nucleotide and amino acid
sequences and the polypeptides encoded thereby are also encompassed
by the embodiments. As used herein the term "fragment" refers to a
portion of a nucleotide sequence of a polynucleotide or a portion
of an amino acid sequence of a polypeptide of the embodiments.
Fragments of a nucleotide sequence may encode protein fragments
that retain the biological activity of the native or corresponding
full-length protein and hence possess pesticidal activity. Thus, it
is acknowledged that some of the polynucleotide and amino acid
sequences of the embodiments can correctly be referred to as both
fragments and mutants.
[0085] It is to be understood that the term "fragment," as it is
used to refer to nucleic acid sequences of the embodiments, also
encompasses sequences that are useful as hybridization probes. This
class of nucleotide sequences generally does not encode fragment
proteins retaining biological activity. Thus, fragments of a
nucleotide sequence may range from at least about 20 nucleotides,
about 50 nucleotides, about 100 nucleotides, and up to the
full-length nucleotide sequence encoding the proteins of the
embodiments.
[0086] A fragment of a nucleotide sequence of the embodiments that
encodes a biologically active portion of a pesticidal protein of
the embodiments will encode at least 15, 25, 30, 50, 100, 200, 250
or 300 contiguous amino acids, or up to the total number of amino
acids present in a pesticidal polypeptide of the embodiments (for
example, 651 amino acids for SEQ ID NO: 3). Thus, it is understood
that the embodiments also encompass polypeptides that are fragments
of the exemplary pesticidal proteins of the embodiments and having
lengths of at least 15, 25, 30, 50, 100, 200, 250 or 300 contiguous
amino acids, or up to the total number of amino acids present in a
pesticidal polypeptide of the embodiments (for example, 651 amino
acids for SEQ ID NO: 3). Fragments of a nucleotide sequence of the
embodiments that are useful as hybridization probes or PCR primers
generally need not encode a biologically active portion of a
pesticidal protein. Thus, a fragment of a nucleic acid of the
embodiments may encode a biologically active portion of a
pesticidal protein, or it may be a fragment that can be used as a
hybridization probe or PCR primer using methods disclosed herein. A
biologically active portion of a pesticidal protein can be prepared
by isolating a portion of one of the nucleotide sequences of the
embodiments, expressing the encoded portion of the pesticidal
protein (e.g., by recombinant expression in vitro), and assessing
the activity of the encoded portion of the pesticidal protein.
[0087] Nucleic acids that are fragments of a nucleotide sequence of
the embodiments comprise at least 16, 20, 50, 75, 100, 150, 200,
250, 300, 350, 400, 450, 500, 600, 700, 800, 850, 900 or 950
nucleotides, or up to the number of nucleotides present in a
nucleotide sequence disclosed herein (for example, 1953 nucleotides
for SEQ ID NO: 4). Particular embodiments envision fragments
derived from (e.g., produced from) a first nucleic acid of the
embodiments, wherein the fragment encodes a truncated toxin having
pesticidal activity. Truncated polypeptides encoded by the
polynucleotide fragments of the embodiments are having pesticidal
activity that is either equivalent to, or improved, relative to the
activity of the corresponding full-length polypeptide encoded by
the first nucleic acid from which the fragment is derived. It is
envisioned that such nucleic acid fragments of the embodiments may
be truncated at the 3' end of the native or corresponding
full-length coding sequence. Nucleic acid fragments may also be
truncated at both the 5' and 3' end of the native or corresponding
full-length coding sequence.
[0088] The term "variants" is used herein to refer to substantially
similar sequences. For nucleotide sequences, conservative variants
include those sequences that, because of the degeneracy of the
genetic code, encode the amino acid sequence of one of the
pesticidal polypeptides of the embodiments. Those having ordinary
skill in the art will readily appreciate that due to the degeneracy
of the genetic code, a multitude of nucleotide sequences encoding
of the present disclosure exist.
[0089] In some embodiments the nucleic acid molecule encoding the
polypeptide is a non-genomic nucleic acid sequence. As used herein
a "non-genomic nucleic acid sequence" or "non-genomic nucleic acid
molecule" or "non-genomic polynucleotide" refers to a nucleic acid
molecule that has one or more change in the nucleic acid sequence
compared to a native or genomic nucleic acid sequence. In some
embodiments the change to a native or genomic nucleic acid molecule
includes but is not limited to: changes in the nucleic acid
sequence due to the degeneracy of the genetic code; codon
optimization of the nucleic acid sequence for expression in plants;
changes in the nucleic acid sequence to introduce at least one
amino acid substitution, insertion, deletion and/or addition
compared to the native or genomic sequence; removal of one or more
intron associated with the genomic nucleic acid sequence; insertion
of one or more heterologous introns; deletion of one or more
upstream or downstream regulatory regions associated with the
genomic nucleic acid sequence; insertion of one or more
heterologous upstream or downstream regulatory regions; deletion of
the 5' and/or 3' untranslated region associated with the genomic
nucleic acid sequence; insertion of a heterologous 5' and/or 3'
untranslated region; and modification of a polyadenylation site. In
some embodiments the non-genomic nucleic acid molecule is a cDNA.
In some embodiments the non-genomic nucleic acid molecule is a
synthetic nucleic acid sequence.
[0090] Where appropriate, a nucleic acid may be optimized for
increased expression in the host organism. Thus, where the host
organism is a plant, the synthetic nucleic acids can be synthesized
using plant-preferred codons for improved expression. See, for
example, Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a
discussion of host-preferred codon usage. For example, although
nucleic acid sequences of the embodiments may be expressed in both
monocotyledonous and dicotyledonous plant species, sequences can be
modified to account for the specific codon preferences and GC
content preferences of monocotyledons or dicotyledons as these
preferences have been shown to differ (Murray et al. (1989) Nucleic
Acids Res. 17:477-498). Thus, the maize-preferred codon for a
particular amino acid may be derived from known gene sequences from
maize. Maize codon usage for 28 genes from maize plants is listed
in Table 4 of Murray, et al., supra. Methods are available in the
art for synthesizing plant-preferred genes. See, for example, U.S.
Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al., (1989)
Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep
37:677-684, 2010, herein incorporated by reference. A Zea maize
codon usage table can be also found at
kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can be
accessed using the www prefix.
[0091] A Glycine max codon usage table is shown in Table 3 and can
also be found at
kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style-
=N, which can be accessed using the www prefix.
[0092] The skilled artisan will further appreciate that changes can
be introduced by mutation of the nucleic acid sequences thereby
leading to changes in the amino acid sequence of the encoded
polypeptides, without altering the biological activity of the
proteins. Thus, variant nucleic acid molecules can be created by
introducing one or more nucleotide substitutions, additions and/or
deletions into the corresponding nucleic acid sequence disclosed
herein, such that one or more amino acid substitutions, additions
or deletions are introduced into the encoded protein. Mutations can
be introduced by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid
sequences are also encompassed by the present disclosure.
[0093] Naturally occurring allelic variants such as these can be
identified with the use of well-known molecular biology techniques,
such as, for example, polymerase chain reaction (PCR) and
hybridization techniques as outlined herein.
[0094] In some embodiments the polynucleotide encoding the
polypeptide of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ
ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:
27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ
ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID
NO: 45 is a non-genomic nucleic acid sequence.
[0095] Variant nucleotide sequences also include synthetically
derived nucleotide sequences, such as those generated, for example,
by using site-directed mutagenesis but which still encode a
pesticidal protein of the embodiments, such as a mutant toxin.
Generally, variants of a particular nucleotide sequence of the
embodiments will have at least about 70%, 75%, 80%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
sequence identity to that particular nucleotide sequence as
determined by sequence alignment programs described elsewhere
herein using default parameters. A variant of a nucleotide sequence
of the embodiments may differ from that sequence by as few as 1-15
nucleotides, as few as 1-10, such as 6-10, as few as 5, as few as
4, 3, 2, or even 1 nucleotide.
[0096] Variants of a particular nucleotide sequence of the
embodiments (i.e., an exemplary nucleotide sequence) can also be
evaluated by comparison of the percent sequence identity between
the polypeptide encoded by a variant nucleotide sequence and the
polypeptide encoded by the reference nucleotide sequence. Thus, for
example, isolated nucleic acids that encode a polypeptide with a
given percent sequence identity to the polypeptides of SEQ ID NO:
3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID
NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21,
SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 41, SEQ ID NO: 43 or SEQ ID NO: 45 are disclosed.
Percent sequence identity between any two polypeptides can be
calculated using sequence alignment programs described elsewhere
herein using default parameters. Where any given pair of
polynucleotides of the embodiments is evaluated by comparison of
the percent sequence identity shared by the two polypeptides they
encode, the percent sequence identity between the two encoded
polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%,
generally at least about 75%, 80%, 85%, at least about 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, or at least about 98%, 99% or more
sequence identity.
[0097] As used herein, the term "variant protein" encompasses
polypeptides that are derived from a native protein by: deletion
(so-called truncation) or addition of one or more amino acids to
the N-terminal and/or C-terminal end of the native protein;
deletion or addition of one or more amino acids at one or more
sites in the native protein; or substitution of one or more amino
acids at one or more sites in the native protein. Accordingly, the
term "variant protein" encompasses biologically active fragments of
a native protein that comprise a sufficient number of contiguous
amino acid residues to retain the biological activity of the native
protein, i.e., to have pesticidal activity. Such pesticidal
activity may be different or improved relative to the native
protein or it may be unchanged, so long as pesticidal activity is
retained.
[0098] Variant proteins encompassed by the embodiments are
biologically active, that is they continue to possess the desired
biological activity of the native protein, that is, pesticidal
activity as described herein. Such variants may result from, for
example, genetic polymorphism or from human manipulation.
Biologically active variants of a native pesticidal protein of the
embodiments will have at least about 60%, 65%, 70%, 75%, 80%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more sequence identity to the amino acid sequence for the
native protein as determined by sequence alignment programs
described elsewhere herein using default parameters. A biologically
active variant of a protein of the embodiments may differ from that
protein by as few as 1-15 amino acid residues, as few as 1-10, such
as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0099] In some embodiment the insecticidal polypeptide has at least
60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the
amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,
SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID
NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43
or SEQ ID NO: 45.
[0100] In some embodiments the polypeptide has a modified physical
property. As used herein, the term "physical property" refers to
any parameter suitable for describing the physical-chemical
characteristics of a protein. As used herein, "physical property of
interest" and "property of interest" are used interchangeably to
refer to physical properties of proteins that are being
investigated and/or modified. Examples of physical properties
include, but are not limited to net surface charge and charge
distribution on the protein surface, net hydrophobicity and
hydrophobic residue distribution on the protein surface, surface
charge density, surface hydrophobicity density, total count of
surface ionizable groups, surface tension, protein size and its
distribution in solution, melting temperature, heat capacity, and
second virial coefficient. Examples of physical properties also
include, but are not limited to solubility, folding, stability, and
digestibility. In some embodiments the polypeptide has increased
digestibility of proteolytic fragments in an insect gut. In some
embodiments the polypeptide has increased stability in an insect
gut. Models for digestion by simulated gastric fluids are known to
one skilled in the art (Fuchs, R. L. and J. D. Astwood. Food
Technology 50: 83-88, 1996; Astwood, J. D., et al Nature
Biotechnology 14: 1269-1273, 1996; Fu T J et al J. Agric Food Chem.
50: 7154-7160, 2002).
[0101] The embodiments further encompass a microorganism that is
transformed with at least one nucleic acid of the embodiments, with
an expression cassette comprising the nucleic acid, or with a
vector comprising the expression cassette. In some embodiments, the
microorganism is one that multiplies on plants. An embodiment of
the disclosure relates to an encapsulated pesticidal protein which
comprises a transformed microorganism capable of expressing at
least one pesticidal protein of the embodiments.
[0102] The embodiments provide pesticidal compositions comprising a
transformed microorganism of the embodiments. In such embodiments,
the transformed microorganism is generally present in the
pesticidal composition in a pesticidally effective amount, together
with a suitable carrier. The embodiments also encompass pesticidal
compositions comprising an isolated protein of the embodiments,
alone or in combination with a transformed organism of the
embodiments and/or an encapsulated pesticidal protein of the
embodiments, in an insecticidally effective amount, together with a
suitable carrier.
[0103] The embodiments further provide a method of increasing
insect target range by using a pesticidal protein of the
embodiments in combination with at least one other or "second"
pesticidal protein. Any pesticidal protein known in the art can be
employed in the methods of the embodiments. Such pesticidal
proteins include, but are not limited to, Bt toxins, protease
inhibitors, .alpha.-amylases, and peroxidases.
[0104] The embodiments also encompass transformed or transgenic
plants comprising at least one nucleotide sequence of the
embodiments. In some embodiments, the plant is stably transformed
with a nucleotide construct comprising at least one nucleotide
sequence of the embodiments operably linked to a promoter that
drives expression in a plant cell. As used herein, the terms
"transformed plant" and "transgenic plant" refer to a plant that
comprises within its genome a heterologous polynucleotide.
Generally, the heterologous polynucleotide is stably integrated
within the genome of a transgenic or transformed plant such that
the polynucleotide is passed on to successive generations. The
heterologous polynucleotide may be integrated into the genome alone
or as part of a recombinant expression cassette.
[0105] It is to be understood that as used herein the term
"transgenic" includes any cell, cell line, callus, tissue, plant
part, or plant the genotype of which has been altered by the
presence of heterologous nucleic acid including those transgenics
initially so altered as well as those created by sexual crosses or
asexual propagation from the initial transgenic. The term
"transgenic" as used herein does not encompass the alteration of
the genome (chromosomal or extra-chromosomal) by conventional plant
breeding methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0106] As used herein, the term "plant" includes whole plants,
plant organs (e.g., leaves, stems, roots, etc.), seeds, plant
cells, and progeny of same. Parts of transgenic plants are within
the scope of the embodiments and comprise, for example, plant
cells, plant protoplasts, plant cell tissue cultures from which
plants can be regenerated, plant calli, plant clumps, and plant
cells that are intact in plants or parts of plants such as embryos,
pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels,
ears, cobs, husks, stalks, roots, root tips, anthers, and the like,
originating in transgenic plants or their progeny previously
transformed with a DNA molecule of the embodiments and therefore
consisting at least in part of transgenic cells. The class of
plants that can be used in the methods of the embodiments is
generally as broad as the class of higher plants amenable to
transformation techniques, including both monocotyledonous and
dicotyledonous plants.
[0107] While the embodiments do not depend on a particular
biological mechanism for increasing the resistance of a plant to a
plant pest, expression of the nucleotide sequences of the
embodiments in a plant can result in the production of the
pesticidal proteins of the embodiments and in an increase in the
resistance of the plant to a plant pest. The plants of the
embodiments find use in agriculture in methods for impacting insect
pests. Certain embodiments provide transformed crop plants, such
as, for example, maize plants, which find use in methods for
impacting insect pests of the plant, such as, for example,
Lepidopteran pests.
[0108] A "subject plant or plant cell" is one in which genetic
alteration, such as transformation, has been affected as to a gene
of interest, or is a plant or plant cell which is descended from a
plant or cell so altered and which comprises the alteration. A
"control" or "control plant" or "control plant cell" provides a
reference point for measuring changes in phenotype of the subject
plant or plant cell.
[0109] A control plant or plant cell may comprise, for example: (a)
a wild-type plant or cell, i.e., of the same genotype as the
starting material for the genetic alteration which resulted in the
subject plant or cell; (b) a plant or plant cell of the same
genotype as the starting material but which has been transformed
with a null construct (i.e., with a construct which has no known
effect on the trait of interest, such as a construct comprising a
marker gene); (c) a plant or plant cell which is a non-transformed
segregant among progeny of a subject plant or plant cell; (d) a
plant or plant cell genetically identical to the subject plant or
plant cell but which is not exposed to conditions or stimuli that
would induce expression of the gene of interest; or (e) the subject
plant or plant cell itself, under conditions in which the gene of
interest is not expressed.
[0110] One of skill in the art will readily acknowledge that
advances in the field of molecular biology such as site-specific
and random mutagenesis, polymerase chain reaction methodologies,
and protein engineering techniques provide an extensive collection
of tools and protocols suitable for use to alter or engineer both
the amino acid sequence and underlying genetic sequences of
proteins of agricultural interest.
[0111] Thus, the proteins of the embodiments may be altered in
various ways including amino acid substitutions, deletions,
truncations, and insertions. Methods for such manipulations are
generally known in the art. For example, amino acid sequence
variants of the pesticidal proteins can be prepared by introducing
mutations into a synthetic nucleic acid (e.g., DNA molecule).
Methods for mutagenesis and nucleic acid alterations are well known
in the art. For example, designed changes can be introduced using
an oligonucleotide-mediated site-directed mutagenesis technique.
See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA
82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382;
U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques
in Molecular Biology (MacMillan Publishing Company, New York), and
the references cited therein.
[0112] The mutagenized nucleotide sequences of the embodiments may
be modified so as to change about 1, 2, 3, 4, 5, 6, 8, 10, 12 or
more of the amino acids present in the primary sequence of the
encoded polypeptide. Alternatively, even more changes from the
native sequence may be introduced such that the encoded protein may
have at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%,
10%, 11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or
20%, 21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more of the
codons altered, or otherwise modified compared to the corresponding
wild-type protein. In the same manner, the encoded protein may have
at least about 1% or 2%, or about 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
11%, 12%, or even about 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%,
21%, 22%, 23%, 24%, or 25%, 30%, 35%, or 40% or more additional
codons compared to the corresponding wild-type protein. It should
be understood that the mutagenized nucleotide sequences of the
embodiments are intended to encompass biologically functional,
equivalent peptides which have pesticidal activity, such as an
improved pesticidal activity as determined by antifeedant
properties against European corn borer larvae. Such sequences may
arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded.
[0113] One of skill in the art would recognize that amino acid
additions and/or substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, charge, size, and the like. Exemplary amino
acid substitution groups that take several of the foregoing
characteristics into consideration are well known to those of skill
in the art and include: arginine and lysine; glutamate and
aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine, and isoleucine.
[0114] Guidance as to appropriate amino acid substitutions that do
not affect biological activity of the protein of interest may be
found in the model of Dayhoff et al. (1978) Atlas of Protein
Sequence and Structure (Natl. Biomed. Res. Found., Washington,
D.C.), herein incorporated by reference. Conservative
substitutions, such as exchanging one amino acid with another
having similar properties, may be made.
[0115] Thus, the genes and nucleotide sequences of the embodiments
include both the naturally occurring sequences and mutant forms.
Likewise, the proteins of the embodiments encompass both naturally
occurring proteins and variations (e.g., truncated polypeptides)
and modified (e.g., mutant) forms thereof. Such variants will
continue to possess the desired pesticidal activity. Obviously, the
mutations that will be made in the nucleotide sequence encoding the
variant must not place the sequence out of reading frame and
generally will not create complementary regions that could produce
secondary mRNA structure. See, EP Patent Application Publication
No. 75,444.
[0116] The deletions, insertions, and substitutions of the protein
sequences encompassed herein are not expected to produce radical
changes in the characteristics of the protein. However, when it is
difficult to predict the exact effect of the substitution,
deletion, or insertion in advance of doing so, one skilled in the
art will appreciate that the effect will be evaluated by routine
screening assays, such as insect-feeding assays. See, for example,
Marrone et al. (1985) J. Econ. Entomol. 78: 290-293 and Czapla and
Lang (1990) J. Econ. Entomol. 83: 2480-2485, herein incorporated by
reference.
[0117] Variant nucleotide sequences and proteins also encompass
sequences and proteins derived from a mutagenic and recombinogenic
procedure such as DNA shuffling. With such a procedure, one or more
different coding sequences can be manipulated to create a new
pesticidal protein possessing the desired properties. In this
manner, libraries of recombinant polynucleotides are generated from
a population of related sequence polynucleotides comprising
sequence regions that have substantial sequence identity and can be
homologously recombined in vitro or in vivo. For example, using
this approach, full-length coding sequences, sequence motifs
encoding a domain of interest, or any fragment of a nucleotide
sequence of the embodiments may be shuffled between the nucleotide
sequences of the embodiments and corresponding portions of other
known Cry nucleotide sequences to obtain a new gene coding for a
protein with an improved property of interest.
[0118] Properties of interest include, but are not limited to,
pesticidal activity per unit of pesticidal protein, protein
stability, and toxicity to non-target species particularly humans,
livestock, and plants and microbes that express the pesticidal
polypeptides of the embodiments. The embodiments are not bound by a
particular shuffling strategy, only that at least one nucleotide
sequence of the embodiments, or part thereof, is involved in such a
shuffling strategy. Shuffling may involve only nucleotide sequences
disclosed herein or may additionally involve shuffling of other
nucleotide sequences known in the art. Strategies for DNA shuffling
are known in the art. See, for example, Stemmer (1994) Proc. Natl.
Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391;
Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al.
(1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.
Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature
391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0119] The nucleotide sequences of the embodiments can also be used
to isolate corresponding sequences from other organisms,
particularly other bacteria, and more particularly other Bacillus
strains. In this manner, methods such as PCR, hybridization, and
the like can be used to identify such sequences based on their
sequence homology to the sequences set forth herein. Sequences that
are selected based on their sequence identity to the entire
sequences set forth herein or to fragments thereof are encompassed
by the embodiments. Such sequences include sequences that are
orthologs of the disclosed sequences. The term "orthologs" refers
to genes derived from a common ancestral gene and which are found
in different species as a result of speciation. Genes found in
different species are considered orthologs when their nucleotide
sequences and/or their encoded protein sequences share substantial
identity as defined elsewhere herein. Functions of orthologs are
often highly conserved among species.
[0120] In a PCR approach, oligonucleotide primers can be designed
for use in PCR reactions to amplify corresponding DNA sequences
from cDNA or genomic DNA extracted from any organism of interest.
Methods for designing PCR primers and PCR cloning are generally
known in the art and are disclosed in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor
Laboratory Press, Plainview, N.Y.), hereinafter "Sambrook". See
also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods
and Applications (Academic Press, New York); Innis and Gelfand,
eds. (1995) PCR Strategies (Academic Press, New York); and Innis
and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New
York). Known methods of PCR include, but are not limited to,
methods using paired primers, nested primers, single specific
primers, degenerate primers, gene-specific primers, vector-specific
primers, partially-mismatched primers, and the like.
[0121] In hybridization techniques, all or part of a known
nucleotide sequence is used as a probe that selectively hybridizes
to other corresponding nucleotide sequences present in a population
of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or
cDNA libraries) from a chosen organism. The hybridization probes
may be genomic DNA fragments, cDNA fragments, RNA fragments, or
other oligonucleotides, and may be labeled with a detectable group
such as .sup.32P or any other detectable marker. Thus, for example,
probes for hybridization can be made by labeling synthetic
oligonucleotides based on the sequences of the embodiments. Methods
for preparation of probes for hybridization and for construction of
cDNA and genomic libraries are generally known in the art and are
disclosed in Sambrook.
[0122] For example, an entire sequence disclosed herein, or one or
more portions thereof, may be used as a probe capable of
specifically hybridizing to corresponding sequences and messenger
RNAs. To achieve specific hybridization under a variety of
conditions, such probes include sequences that are unique to the
sequences of the embodiments and are generally at least about 10 or
20 nucleotides in length. Such probes may be used to amplify
corresponding Cry sequences from a chosen organism by PCR. This
technique may be used to isolate additional coding sequences from a
desired organism or as a diagnostic assay to determine the presence
of coding sequences in an organism. Hybridization techniques
include hybridization screening of plated DNA libraries (either
plaques or colonies; see, for example, Sambrook).
[0123] Hybridization of such sequences may be carried out under
stringent conditions. The term "stringent conditions" or "stringent
hybridization conditions" as used herein refers to conditions under
which a probe will hybridize to its target sequence to a detectably
greater degree than to other sequences (e.g., at least 2-fold,
5-fold, or 10-fold over background). Stringent conditions are
sequence-dependent and will be different in different
circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences that are 100%
complementary to the probe can be identified (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some
mismatching in sequences so that lower degrees of similarity are
detected (heterologous probing). Generally, a probe is less than
about 1000 or 500 nucleotides in length.
[0124] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide. Exemplary low stringency conditions
include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37.degree.
C., and a wash in 1.times. to 2.times.SSC (20.times.SSC=3.0 M
NaCl/0.3 M trisodium citrate) at 50 to 55.degree. C. Exemplary
moderate stringency conditions include hybridization in 40 to 45%
formamide, 1.0 M NaCl, 1% SDS at 37 C, and a wash in 0.5.times. to
1.times.SSC at 55 to 60.degree. C. Exemplary high stringency
conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS
at 37.degree. C., and a final wash in 0.1.times.SSC at 60 to
65.degree. C. for at least about 20 minutes. Optionally, wash
buffers may comprise about 0.1% to about 1% SDS. The duration of
hybridization is generally less than about 24 hours, usually about
4 to about 12 hours.
[0125] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b) "comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0126] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0127] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0128] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent sequence
identity between any two sequences can be accomplished using a
mathematical algorithm. Non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller (1988) CABIOS
4:11-17; the local alignment algorithm of Smith et al. (1981) Adv.
Appl. Math. 2:482; the global alignment algorithm of Needleman and
Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local
alignment method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990)
Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.
[0129] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics
Software Package, Version 10 (available from Accelrys Inc., 9685
Scranton Road, San Diego, Calif., USA). Alignments using these
programs can be performed using the default parameters. The CLUSTAL
program is well described by Higgins et al. (1988) Gene 73:237-244
(1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al.
(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS
8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.
The ALIGN program is based on the algorithm of Myers and Miller
(1988) supra. A PAM120 weight residue table, a gap length penalty
of 12, and a gap penalty of 4 can be used with the ALIGN program
when comparing amino acid sequences. The BLAST programs of Altschul
et al (1990) J. Mol. Biol. 215:403 are based on the algorithm of
Karlin and Altschul (1990) supra. BLAST nucleotide searches can be
performed with the BLASTN program, score=100, wordlength=12, to
obtain nucleotide sequences homologous to a nucleotide sequence
encoding a protein of the embodiments. BLAST protein searches can
be performed with the BLASTX program, score=50, wordlength=3, to
obtain amino acid sequences homologous to a protein or polypeptide
of the embodiments. To obtain gapped alignments for comparison
purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25:3389.
Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an
iterated search that detects distant relationships between
molecules. See Altschul et al. (1997) supra. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. See the National Center for Biotechnology
Information website on the world wide web at ncbi.hlm.nih.gov.
Alignment may also be performed manually by inspection.
[0130] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to the residues in the two sequences that are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity". Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0131] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0132] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70%. 80%, 90%, or 95% or more sequence identity when
compared to a reference sequence using one of the alignment
programs described using standard parameters. One of skill in the
art will recognize that these values can be appropriately adjusted
to determine corresponding identity of proteins encoded by two
nucleotide sequences by taking into account codon degeneracy, amino
acid similarity, reading frame positioning, and the like.
Substantial identity of amino acid sequences for these purposes
generally means sequence identity of at least 60%, 70%, 80%, 90%,
or 95% or more sequence identity.
[0133] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the T.sub.m for the
specific sequence at a defined ionic strength and pH. However,
stringent conditions encompass temperatures in the range of about
1.degree. C. to about 20.degree. C. lower than the T.sub.m,
depending upon the desired degree of stringency as otherwise
qualified herein. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0134] (e)(ii) The term "substantial identity" in the context of a
peptide indicates that a peptide comprises a sequence with at least
70%, 80%, 85%, 90%, 95%, or more sequence identity to a reference
sequence over a specified comparison window. Optimal alignment for
these purposes can be conducted using the global alignment
algorithm of Needleman and Wunsch (1970) supra. An indication that
two peptide sequences are substantially identical is that one
peptide is immunologically reactive with antibodies raised against
the second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. Peptides that are "substantially
similar" share sequences as noted above except that residue
positions that are not identical may differ by conservative amino
acid changes.
[0135] The use of the term "nucleotide constructs" herein is not
intended to limit the embodiments to nucleotide constructs
comprising DNA. Those of ordinary skill in the art will recognize
that nucleotide constructs, particularly polynucleotides and
oligonucleotides composed of ribonucleotides and combinations of
ribonucleotides and deoxyribonucleotides, may also be employed in
the methods disclosed herein. The nucleotide constructs, nucleic
acids, and nucleotide sequences of the embodiments additionally
encompass all complementary forms of such constructs, molecules,
and sequences. Further, the nucleotide constructs, nucleotide
molecules, and nucleotide sequences of the embodiments encompass
all nucleotide constructs, molecules, and sequences which can be
employed in the methods of the embodiments for transforming plants
including, but not limited to, those comprised of
deoxyribonucleotides, ribonucleotides, and combinations thereof.
Such deoxyribonucleotides and ribonucleotides include both
naturally occurring molecules and synthetic analogues. The
nucleotide constructs, nucleic acids, and nucleotide sequences of
the embodiments also encompass all forms of nucleotide constructs
including, but not limited to, single-stranded forms,
double-stranded forms, hairpins, stem-and-loop structures, and the
like.
[0136] A further embodiment relates to a transformed organism such
as an organism selected from the group consisting of plant and
insect cells, bacteria, yeast, baculovirus, protozoa, nematodes,
and algae. The transformed organism comprises: a DNA molecule of
the embodiments, an expression cassette comprising the said DNA
molecule, or a vector comprising the said expression cassette,
which may be stably incorporated into the genome of the transformed
organism.
[0137] The sequences of the embodiments are provided in DNA
constructs for expression in the organism of interest. The
construct will include 5' and 3' regulatory sequences operably
linked to a sequence of the embodiments. The term "operably linked"
as used herein refers to a functional linkage between a promoter
and a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA sequence corresponding to the
second sequence. Generally, operably linked means that the nucleic
acid sequences being linked are contiguous and, where necessary to
join two protein coding regions, contiguous and in the same reading
frame. The construct may additionally contain at least one
additional gene to be cotransformed into the organism.
Alternatively, the additional gene(s) can be provided on multiple
DNA constructs.
[0138] Such a DNA construct is provided with a plurality of
restriction sites for insertion of the Cry toxin sequence to be
under the transcriptional regulation of the regulatory regions. The
DNA construct may additionally contain selectable marker genes.
[0139] The DNA construct will include in the 5' to 3' direction of
transcription: a transcriptional and translational initiation
region (i.e., a promoter), a DNA sequence of the embodiments, and a
transcriptional and translational termination region (i.e.,
termination region) functional in the organism serving as a host.
The transcriptional initiation region (i.e., the promoter) may be
native, analogous, foreign or heterologous to the host organism
and/or to the sequence of the embodiments. Additionally, the
promoter may be the natural sequence or alternatively a synthetic
sequence. The term "foreign" as used herein indicates that the
promoter is not found in the native organism into which the
promoter is introduced. Where the promoter is "foreign" or
"heterologous" to the sequence of the embodiments, it is intended
that the promoter is not the native or naturally occurring promoter
for the operably linked sequence of the embodiments. As used
herein, a chimeric gene comprises a coding sequence operably linked
to a transcription initiation region that is heterologous to the
coding sequence. Where the promoter is a native or natural
sequence, the expression of the operably linked sequence is altered
from the wild-type expression, which results in an alteration in
phenotype.
[0140] The termination region may be native with the
transcriptional initiation region, may be native with the operably
linked DNA sequence of interest, may be native with the plant host,
or may be derived from another source (i.e., foreign or
heterologous to the promoter, the sequence of interest, the plant
host, or any combination thereof).
[0141] Convenient termination regions are available from the
Ti-plasmid of A. tumefaciens, such as the octopine synthase and
nopaline synthase termination regions. See also Guerineau et al.
(1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell
64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et
al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene
91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903;
and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0142] Where appropriate, a nucleic acid may be optimized for
increased expression in the host organism. Thus, where the host
organism is a plant, the synthetic nucleic acids can be synthesized
using plant-preferred codons for improved expression. See, for
example, Campbell and Gown (1990) Plant Physiol. 92:1-11 for a
discussion of host-preferred codon usage. For example, although
nucleic acid sequences of the embodiments may be expressed in both
monocotyledonous and dicotyledonous plant species, sequences can be
modified to account for the specific codon preferences and GC
content preferences of monocotyledons or dicotyledons as these
preferences have been shown to differ (Murray et al. (1989) Nucleic
Acids Res. 17:477-498). Thus, the maize-preferred codon for a
particular amino acid may be derived from known gene sequences from
maize. Maize codon usage for 28 genes from maize plants is listed
in Table 4 of Murray et al., supra. Methods are available in the
art for synthesizing plant-preferred genes. See, for example, U.S.
Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989)
Nucleic Acids Res. 17:477-498, herein incorporated by
reference.
[0143] Additional sequence modifications are known to enhance gene
expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon-intron
splice site signals, transposon-like repeats, and other
well-characterized sequences that may be deleterious to gene
expression. The GC content of the sequence may be adjusted to
levels average for a given cellular host, as calculated by
reference to known genes expressed in the host cell. The term "host
cell" as used herein refers to a cell which contains a vector and
supports the replication and/or expression of the expression vector
is intended. Host cells may be prokaryotic cells such as E. coli,
or eukaryotic cells such as yeast, insect, amphibian, or mammalian
cells, or monocotyledonous or dicotyledonous plant cells. An
example of a monocotyledonous host cell is a maize host cell. When
possible, the sequence is modified to avoid predicted hairpin
secondary mRNA structures.
[0144] The expression cassettes may additionally contain 5' leader
sequences. Such leader sequences can act to enhance translation.
Translation leaders are known in the art and include: picornavirus
leaders, for example, EMCV leader (Encephalomyocarditis 5'
noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci.
USA 86: 6126-6130); potyvirus leaders, for example, TEV leader
(Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238),
MDMV leader (Maize Dwarf Mosaic Virus), human immunoglobulin
heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature
353: 90-94); untranslated leader from the coat protein mRNA of
alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:
622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989)
in Molecular Biology of RNA, ed. Cech (Liss, New York), pp.
237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et
al. (1991) Virology 81: 382-385). See also, Della-Cioppa et al.
(1987) Plant Physiol. 84: 965-968.
[0145] In preparing the expression cassette, the various DNA
fragments may be manipulated so as to provide for the DNA sequences
in the proper orientation and, as appropriate, in the proper
reading frame. Toward this end, adapters or linkers may be employed
to join the DNA fragments or other manipulations may be involved to
provide for convenient restriction sites, removal of superfluous
DNA, removal of restriction sites, or the like. For this purpose,
in vitro mutagenesis, primer repair, restriction, annealing,
resubstitutions, e.g., transitions and transversions, may be
involved.
[0146] A number of promoters can be used in the practice of the
embodiments. The promoters can be selected based on the desired
outcome. The nucleic acids can be combined with constitutive,
tissue-preferred, inducible, or other promoters for expression in
the host organism. Suitable constitutive promoters for use in a
plant host cell include, for example, the core promoter of the
Rsyn7 promoter and other constitutive promoters disclosed in WO
99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter
(Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et
al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al.
(1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992)
Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theor.
Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J.
3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like.
Other constitutive promoters include, for example, those discussed
in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0147] Depending on the desired outcome, it may be beneficial to
express the gene from an inducible promoter. Of particular interest
for regulating the expression of the nucleotide sequences of the
embodiments in plants are wound-inducible promoters. Such
wound-inducible promoters, may respond to damage caused by insect
feeding, and include potato proteinase inhibitor (pin II) gene
(Ryan (1990) Ann. Rev. Phytopath. 28: 425-449; Duan et al. (1996)
Nature Biotechnology 14: 494-498); wun1 and wun2, U.S. Pat. No.
5,428,148; win1 and win2 (Stanford et al. (1989) Mol. Gen. Genet.
215: 200-208); systemin (McGurl et al. (1992) Science 225:
1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22:
783-792; Eckelkamp et al. (1993) FEBS Letters 323: 73-76); MPI gene
(Corderok et al. (1994) Plant J. 6(2): 141-150); and the like,
herein incorporated by reference.
[0148] Additionally, pathogen-inducible promoters may be employed
in the methods and nucleotide constructs of the embodiments. Such
pathogen-inducible promoters include those from
pathogenesis-related proteins (PR proteins), which are induced
following infection by a pathogen; e.g., PR proteins, SAR proteins,
beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et
al. (1983) Neth. J. Plant Pathol. 89: 245-254; Uknes et al. (1992)
Plant Cell 14: 645-656; and Van Loon (1985) Plant Mol. Virol. 4:
111-116. See also WO 99/43819, herein incorporated by
reference.
[0149] Of interest are promoters that are expressed locally at or
near the site of pathogen infection. See, for example, Marineau et
al. (1987) Plant Mol. Biol. 9:335-342; Matton et al. (1989)
Molecular Plant-Microbe Interactions 2:325-331; Somsisch et al.
(1986) Proc. Natl. Acad. Sci. USA 83:2427-2430; Somsisch et al.
(1988) Mol. Gen. Genet. 2:93-98; and Yang (1996) Proc. Natl. Acad.
Sci. USA 93:14972-14977. See also, Chen et al. (1996) Plant J.
10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci. USA
91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et
al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386
(nematode-inducible); and the references cited therein. Of
particular interest is the inducible promoter for the maize PRms
gene, whose expression is induced by the pathogen Fusarium
moniliforme (see, for example, Cordero et al. (1992) Physiol. Mol.
Plant Path. 41:189-200).
[0150] Chemical-regulated promoters can be used to modulate the
expression of a gene in a plant through the application of an
exogenous chemical regulator. Depending upon the objective, the
promoter may be a chemical-inducible promoter, where application of
the chemical induces gene expression, or a chemical-repressible
promoter, where application of the chemical represses gene
expression. Chemical-inducible promoters are known in the art and
include, but are not limited to, the maize In2-2 promoter, which is
activated by benzenesulfonamide herbicide safeners, the maize GST
promoter, which is activated by hydrophobic electrophilic compounds
that are used as pre-emergent herbicides, and the tobacco PR-1a
promoter, which is activated by salicylic acid. Other
chemical-regulated promoters of interest include steroid-responsive
promoters (see, for example, the glucocorticoid-inducible promoter
in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425
and McNellis et al. (1998) Plant J. 14(2):247-257) and
tetracycline-inducible and tetracycline-repressible promoters (see,
for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and
U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by
reference.
[0151] Tissue-preferred promoters can be utilized to target
enhanced pesticidal protein expression within a particular plant
tissue. Tissue-preferred promoters include those discussed in
Yamamoto et al. (1997) Plant J. 12(2)255-265; Kawamata et al.
(1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol.
Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.
6(2):157-168; Rinehart et al. (1996) Plant Physiol.
112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.
112(2):525-535; Canevascini et al. (1996) Plant Physiol.
112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol.
35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196;
Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138; Matsuoka et
al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and
Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters
can be modified, if necessary, for weak expression.
[0152] Leaf-preferred promoters are known in the art. See, for
example, Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al.
(1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell
Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18;
Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka
et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
[0153] Root-preferred or root-specific promoters are known and can
be selected from the many available from the literature or isolated
de novo from various compatible species. See, for example, Hire et
al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific
glutamine synthetase gene); Keller and Baumgartner (1991) Plant
Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8
gene of French bean); Sanger et al. (1990) Plant Mol. Biol.
14(3):433-443 (root-specific promoter of the mannopine synthase
(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991)
Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic
glutamine synthetase (GS), which is expressed in roots and root
nodules of soybean). See also Bogusz et al. (1990) Plant Cell
2(7):633-641, where two root-specific promoters isolated from
hemoglobin genes from the nitrogen-fixing nonlegume Parasponia
andersonii and the related non-nitrogen-fixing nonlegume Trema
tomentosa are described. The promoters of these genes were linked
to a .beta.-glucuronidase reporter gene and introduced into both
the nonlegume Nicotiana tabacum and the legume Lotus comiculatus,
and in both instances root-specific promoter activity was
preserved. Leach and Aoyagi (1991) describe their analysis of the
promoters of the highly expressed rolC and rolD root-inducing genes
of Agrobacterium rhizogenes (see Plant Science (Limerick)
79(1):69-76). They concluded that enhancer and tissue-preferred DNA
determinants are dissociated in those promoters. Teen et al. (1989)
used gene fusion to lacZ to show that the Agrobacterium T-DNA gene
encoding octopine synthase is especially active in the epidermis of
the root tip and that the TR2' gene is root specific in the intact
plant and stimulated by wounding in leaf tissue, an especially
desirable combination of characteristics for use with an
insecticidal or larvicidal gene (see EMBO J. 8(2):343-350). The
TR1' gene fused to nptII (neomycin phosphotransferase II) showed
similar characteristics. Additional root-preferred promoters
include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant
Mol. Biol. 29(4):759-772); and rolB promoter (Capana et al. (1994)
Plant Mol. Biol. 25(4):681-691. See also U.S. Pat. Nos. 5,837,876;
5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and
5,023,179.
[0154] "Seed-preferred" promoters include both "seed-specific"
promoters (those promoters active during seed development such as
promoters of seed storage proteins) as well as "seed-germinating"
promoters (those promoters active during seed germination). See
Thompson et al. (1989) BioEssays 10:108, herein incorporated by
reference. Such seed-preferred promoters include, but are not
limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa
zein); and milps (myo-inositol-1-phosphate synthase) (see U.S. Pat.
No. 6,225,529, herein incorporated by reference). Gamma-zein and
Glob-1 are endosperm-specific promoters. For dicots, seed-specific
promoters include, but are not limited to, bean .beta.-phaseolin,
napin, .beta.-conglycinin, soybean lectin, cruciferin, and the
like. For monocots, seed-specific promoters include, but are not
limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein,
waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO
00/12733, where seed-preferred promoters from end1 and end2 genes
are disclosed; herein incorporated by reference. A promoter that
has "preferred" expression in a particular tissue is expressed in
that tissue to a greater degree than in at least one other plant
tissue. Some tissue-preferred promoters show expression almost
exclusively in the particular tissue.
[0155] Where low level expression is desired, weak promoters will
be used. Generally, the term "weak promoter" as used herein refers
to a promoter that drives expression of a coding sequence at a low
level. By low level expression at levels of about 1/1000
transcripts to about 1/100,000 transcripts to about 1/500,000
transcripts is intended. Alternatively, it is recognized that the
term "weak promoters" also encompasses promoters that drive
expression in only a few cells and not in others to give a total
low level of expression. Where a promoter drives expression at
unacceptably high levels, portions of the promoter sequence can be
deleted or modified to decrease expression levels.
[0156] Such weak constitutive promoters include, for example the
core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.
6,072,050), the core 35S CaMV promoter, and the like. Other
constitutive promoters include, for example, those disclosed in
U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597;
5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611; herein
incorporated by reference.
[0157] Generally, the expression cassette will comprise a
selectable marker gene for the selection of transformed cells.
Selectable marker genes are utilized for the selection of
transformed cells or tissues. Marker genes include genes encoding
antibiotic resistance, such as those encoding neomycin
phosphotransferase II (NEO) and hygromycin phosphotransferase
(HPT), as well as genes conferring resistance to herbicidal
compounds, such as glufosinate ammonium, bromoxynil,
imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional
examples of suitable selectable marker genes include, but are not
limited to, genes encoding resistance to chloramphenicol (Herrera
Estrella et al. (1983) EMBO J. 2:987-992); methotrexate (Herrera
Estrella et al. (1983) Nature 303:209-213; and Meijer et al. (1991)
Plant Mol. Biol. 16:807-820); streptomycin (Jones et al. (1987)
Mol. Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard et al.
(1996) Transgenic Res. 5:131-137); bleomycin (Hille et al. (1990)
Plant Mol. Biol. 7:171-176); sulfonamide (Guerineau et al. (1990)
Plant Mol. Biol. 15:127-136); bromoxynil (Stalker et al. (1988)
Science 242:419-423); glyphosate (Shaw et al. (1986) Science
233:478-481; and U.S. Pat. Nos. 7,709,702; and 7,462,481);
phosphinothricin (DeBlock et al. (1987) EMBO J. 6:2513-2518). See
generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511;
Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:
6314-6318; Yao et al. (1992) Cell 171: 63-72; Reznikoff (1992) Mol.
Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp.
177-220; Hu et al. (1987) Cell 148: 555-566; Brown et al. (1987)
Cell 149: 603-612; Figge et al. (1988) Cell 152: 713-722; Deuschle
et al. (1989) Proc. Natl. Acad. Sci. USA 86: 5400-5404; Fuerst et
al. (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et
al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis,
University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad.
Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:
3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:
3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:
5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653;
Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162;
Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:
1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104;
Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.
(1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al.
(1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al.
(1985) Handbook of Experimental Pharmacology, Vol. 78
(Springer-Verlag, Berlin); and Gill et al. (1988) Nature 334:
721-724. Such disclosures are herein incorporated by reference.
[0158] The above list of selectable marker genes is not meant to be
limiting. Any selectable marker gene can be used in the
embodiments.
[0159] The methods of the embodiments involve introducing a
polypeptide or polynucleotide into a plant. "Introducing" is
intended to mean presenting to the plant the polynucleotide or
polypeptide in such a manner that the sequence gains access to the
interior of a cell of the plant. The methods of the embodiments do
not depend on a particular method for introducing a polynucleotide
or polypeptide into a plant, only that the polynucleotide or
polypeptides gains access to the interior of at least one cell of
the plant. Methods for introducing polynucleotide or polypeptides
into plants are known in the art including, but not limited to,
stable transformation methods, transient transformation methods,
and virus-mediated methods.
[0160] "Stable transformation" is intended to mean that the
nucleotide construct introduced into a plant integrates into the
genome of the plant and is capable of being inherited by the
progeny thereof. "Transient transformation" is intended to mean
that a polynucleotide is introduced into the plant and does not
integrate into the genome of the plant or a polypeptide is
introduced into a plant.
[0161] Transformation protocols as well as protocols for
introducing nucleotide sequences into plants may vary depending on
the type of plant or plant cell, i.e., monocot or dicot, targeted
for transformation. Suitable methods of introducing nucleotide
sequences into plant cells and subsequent insertion into the plant
genome include microinjection (Crossway et al. (1986) Biotechniques
4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad.
Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation
(U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer
(Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic
particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050;
5,879,918; 5,886,244; and U.S. Pat. No. 5,932,782; Tomes et al.
(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental
Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and
McCabe et al. (1988) Biotechnology 6: 923-926); and LecI
transformation (WO 00/28058). For potato transformation see Tu et
al. (1998) Plant Molecular Biology 37: 829-838 and Chong et al.
(2000) Transgenic Research 9: 71-78. Additional transformation
procedures can be found in Weissinger et al. (1988) Ann. Rev.
Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and
Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol.
87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:
923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev.
Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl.
Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8:
736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:
4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563
(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein
et al. (1988) Plant Physiol. 91: 440-444 (maize); Fromm et al.
(1990) Biotechnology 8: 833-839 (maize); Hooykaas-Van Slogteren et
al. (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369
(cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:
5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental
Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New
York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell
Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.
84: 560-566 (whisker-mediated transformation); D'Halluin et al.
(1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993)
Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals
of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature
Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens);
all of which are herein incorporated by reference.
[0162] In specific embodiments, the sequences of the embodiments
can be provided to a plant using a variety of transient
transformation methods. Such transient transformation methods
include, but are not limited to, the introduction of the Cry toxin
protein or variants and fragments thereof directly into the plant
or the introduction of the Cry toxin transcript into the plant.
Such methods include, for example, microinjection or particle
bombardment. See, for example, Crossway et al. (1986) Mol. Gen.
Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58;
Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush
et al. (1994) The Journal of Cell Science 107: 775-784, all of
which are herein incorporated by reference. Alternatively, the Cry
toxin polynucleotide can be transiently transformed into the plant
using techniques known in the art. Such techniques include viral
vector system and the precipitation of the polynucleotide in a
manner that precludes subsequent release of the DNA. Thus,
transcription from the particle-bound DNA can occur, but the
frequency with which it is released to become integrated into the
genome is greatly reduced. Such methods include the use of
particles coated with polyethylimine (PEI; Sigma #P3143).
[0163] Methods are known in the art for the targeted insertion of a
polynucleotide at a specific location in the plant genome. In one
embodiment, the insertion of the polynucleotide at a desired
genomic location is achieved using a site-specific recombination
system. See, for example, WO99/25821, WO99/25854, WO99/25840,
WO99/25855, and WO99/25853, all of which are herein incorporated by
reference. Briefly, the polynucleotide of the embodiments can be
contained in transfer cassette flanked by two non-identical
recombination sites. The transfer cassette is introduced into a
plant have stably incorporated into its genome a target site which
is flanked by two non-identical recombination sites that correspond
to the sites of the transfer cassette. An appropriate recombinase
is provided and the transfer cassette is integrated at the target
site. The polynucleotide of interest is thereby integrated at a
specific chromosomal position in the plant genome.
[0164] The cells that have been transformed may be grown into
plants in accordance with conventional ways. See, for example,
McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants
may then be grown, and either pollinated with the same transformed
strain or different strains, and the resulting hybrid having
constitutive or inducible expression of the desired phenotypic
characteristic identified. Two or more generations may be grown to
ensure that expression of the desired phenotypic characteristic is
stably maintained and inherited and then seeds harvested to ensure
that expression of the desired phenotypic characteristic has been
achieved.
[0165] The nucleotide sequences of the embodiments may be provided
to the plant by contacting the plant with a virus or viral nucleic
acids. Generally, such methods involve incorporating the nucleotide
construct of interest within a viral DNA or RNA molecule. It is
recognized that the recombinant proteins of the embodiments may be
initially synthesized as part of a viral polyprotein, which later
may be processed by proteolysis in vivo or in vitro to produce the
desired pesticidal protein. It is also recognized that such a viral
polyprotein, comprising at least a portion of the amino acid
sequence of a pesticidal protein of the embodiments, may have the
desired pesticidal activity. Such viral polyproteins and the
nucleotide sequences that encode for them are encompassed by the
embodiments. Methods for providing plants with nucleotide
constructs and producing the encoded proteins in the plants, which
involve viral DNA or RNA molecules are known in the art. See, for
example, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367;
and 5,316,931; herein incorporated by reference.
[0166] The embodiments further relate to plant-propagating material
of a transformed plant of the embodiments including, but not
limited to, seeds, tubers, corms, bulbs, leaves, and cuttings of
roots and shoots.
[0167] The embodiments may be used for transformation of any plant
species, including, but not limited to, monocots and dicots.
Examples of plants of interest include, but are not limited to,
corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),
particularly those Brassica species useful as sources of seed oil,
alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale
cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,
pearl millet (Pennisetum glaucum), proso millet (Panicum
miliaceum), foxtail millet (Setaria italica), finger millet
(Eleusine coracana)), sunflower (Helianthus annuus), safflower
(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine
max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum),
peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium
hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot
esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),
pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa
(Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea americana), fig (Ficus casica), guava (Psidium
guajava), mango (Mangifera indica), olive (Olea europaea), papaya
(Carica papaya), cashew (Anacardium occidentale), macadamia
(Macadamia integrifolia), almond (Prunus amygdalus), sugar beets
(Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers.
[0168] Vegetables include tomatoes (Lycopersicon esculentum),
lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris),
lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members
of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C.
cantalupensis), and musk melon (C. melo). Ornamentals include
azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),
hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa
spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida),
carnation (Dianthus caryophyllus), poinsettia (Euphorbia
pulcherrima), and chrysanthemum. Conifers that may be employed in
practicing the embodiments include, for example, pines such as
loblolly pine (Pinus taeda), slash pine (Pinus effiotii), ponderosa
pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and
Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii);
Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca);
redwood (Sequoia sempervirens); true firs such as silver fir (Abies
amabilis) and balsam fir (Abies balsamea); and cedars such as
Western red cedar (Thuja plicata) and Alaska yellow-cedar
(Chamaecyparis nootkatensis). Plants of the embodiments include
crop plants, including, but not limited to: corn, alfalfa,
sunflower, Brassica spp., soybean, cotton, safflower, peanut,
sorghum, wheat, millet, tobacco, sugarcane, etc.
[0169] Turfgrasses include, but are not limited to: annual
bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canada
bluegrass (Poa compressa); Chewings fescue (Festuca rubra);
colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis
palustris); crested wheatgrass (Agropyron desertorum); fairway
wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia);
Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis
glomerata); perennial ryegrass (Lolium perenne); red fescue
(Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa
trivialis); sheep fescue (Festuca ovina); smooth bromegrass (Bromus
inermis); tall fescue (Festuca arundinacea); timothy (Phleum
pratense); velvet bentgrass (Agrostis canina); weeping alkaligrass
(Puccineffia distans); western wheatgrass (Agropyron smithii);
Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum
secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum
notatum); carpet grass (Axonopus affinis); centipede grass
(Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum);
seashore paspalum (Paspalum vaginatum); blue gramma (Bouteloua
gracilis); buffalo grass (Buchloe dactyloids); sideoats gramma
(Bouteloua curtipendula).
[0170] Plants of interest include grain plants that provide seeds
of interest, oil-seed plants, and leguminous plants. Seeds of
interest include grain seeds, such as corn, wheat, barley, rice,
sorghum, rye, millet, etc. Oil-seed plants include cotton, soybean,
safflower, sunflower, Brassica, maize, alfalfa, palm, coconut,
flax, castor, olive etc. Leguminous plants include beans and peas.
Beans include guar, locust bean, fenugreek, soybean, garden beans,
cowpea, mung bean, lima bean, fava bean, lentils, chickpea,
etc.
[0171] In certain embodiments the nucleic acid sequences of the
embodiments can be stacked with any combination of polynucleotide
sequences of interest in order to create plants with a desired
phenotype. For example, the polynucleotides of the embodiments may
be stacked with any other polynucleotides encoding polypeptides
having pesticidal and/or insecticidal activity, such as other Bt
toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450;
5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene
48:109), pentin (described in U.S. Pat. No. 5,981,722) and the
like. The combinations generated can also include multiple copies
of any one of the polynucleotides of interest. The polynucleotides
of the embodiments can also be stacked with any other gene or
combination of genes to produce plants with a variety of desired
trait combinations including but not limited to traits desirable
for animal feed such as high oil genes (e.g., U.S. Pat. No.
6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat.
Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,049); barley high
lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106; and
WO 98/20122) and high methionine proteins (Pedersen et al. (1986)
J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and
Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased
digestibility (e.g., modified storage proteins (U.S. Pat. No.
6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087), the
disclosures of which are herein incorporated by reference.
[0172] The polynucleotides of the embodiments can also be stacked
with traits desirable for disease or herbicide resistance (e.g.,
fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones et al. (1994)
Science 266:789; Martin et al. (1993) Science 262: 1432; and
Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS)
mutants that lead to herbicide resistance such as the S4 and/or Hra
mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene and GAT gene as disclosed in U.S. Pat. Nos.
7,709,702; and 7,462,481; and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)); and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.
170: 5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)), the disclosures of which are herein incorporated by
reference. One could also combine the polynucleotides of the
embodiments with polynucleotides providing agronomic traits such as
male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,
flowering time, or transformation technology traits such as cell
cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364;
WO 99/25821), the disclosures of which are herein incorporated by
reference.
[0173] In some embodiment the stacked trait may be a trait or event
that has received regulatory approval which are well known to one
skilled in the art and can be found at the Center for Environmental
Risk Assessment (cera-gmc.org/?action=gm_crop_database, which can
be accessed using the www prefix) and at the International Service
for the Acquisition of Agri-Biotech Applications
isaaa.org/gmapprovaldatabase/default.asp, which can be accessed
using the www prefix).
[0174] These stacked combinations can be created by any method
including but not limited to cross breeding plants by any
conventional or TOPCROSS.RTM. methodology, or genetic
transformation. If the traits are stacked by genetically
transforming the plants, the polynucleotide sequences of interest
can be combined at any time and in any order. For example, a
transgenic plant comprising one or more desired traits can be used
as the target to introduce further traits by subsequent
transformation. The traits can be introduced simultaneously in a
co-transformation protocol with the polynucleotides of interest
provided by any combination of transformation cassettes. For
example, if two sequences will be introduced, the two sequences can
be contained in separate transformation cassettes (trans) or
contained on the same transformation cassette (cis). Expression of
the sequences can be driven by the same promoter or by different
promoters. In certain cases, it may be desirable to introduce a
transformation cassette that will suppress the expression of the
polynucleotide of interest. This may be combined with any
combination of other suppression cassettes or overexpression
cassettes to generate the desired combination of traits in the
plant. It is further recognized that polynucleotide sequences can
be stacked at a desired genomic location using a site-specific
recombination system. See, for example, WO99/25821, WO99/25854,
WO99/25840, WO99/25855, and WO99/25853, all of which are herein
incorporated by reference.
[0175] Compositions of the embodiments find use in protecting
plants, seeds, and plant products in a variety of ways. For
example, the compositions can be used in a method that involves
placing an effective amount of the pesticidal composition in the
environment of the pest by a procedure selected from the group
consisting of spraying, dusting, broadcasting, or seed coating.
[0176] Before plant propagation material (fruit, tuber, bulb, corm,
grains, seed), but especially seed, is sold as a commercial
product, it is customarily treated with a protectant coating
comprising herbicides, insecticides, fungicides, bactericides,
nematicides, molluscicides, or mixtures of several of these
preparations, if desired together with further carriers,
surfactants, or application-promoting adjuvants customarily
employed in the art of formulation to provide protection against
damage caused by bacterial, fungal, or animal pests. In order to
treat the seed, the protectant coating may be applied to the seeds
either by impregnating the tubers or grains with a liquid
formulation or by coating them with a combined wet or dry
formulation. In addition, in special cases, other methods of
application to plants are possible, e.g., treatment directed at the
buds or the fruit.
[0177] The plant seed of the embodiments comprising a nucleotide
sequence encoding a pesticidal protein of the embodiments may be
treated with a seed protectant coating comprising a seed treatment
compound, such as, for example, captan, carboxin, thiram,
methalaxyl, pirimiphos-methyl, and others that are commonly used in
seed treatment. In one embodiment, a seed protectant coating
comprising a pesticidal composition of the embodiments is used
alone or in combination with one of the seed protectant coatings
customarily used in seed treatment.
[0178] It is recognized that the genes encoding the pesticidal
proteins can be used to transform insect pathogenic organisms. Such
organisms include baculovirus, fungi, protozoa, bacteria, and
nematodes.
[0179] A gene encoding a pesticidal protein of the embodiments may
be introduced via a suitable vector into a microbial host, and said
host applied to the environment, or to plants or animals. The term
"introduced" in the context of inserting a nucleic acid into a
cell, means "transfection" or "transformation" or "transduction"
and includes reference to the incorporation of a nucleic acid into
a eukaryotic or prokaryotic cell where the nucleic acid may be
incorporated into the genome of the cell (e.g., chromosome,
plasmid, plastid, or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0180] Microorganism hosts that are known to occupy the
"phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or
rhizoplana) of one or more crops of interest may be selected. These
microorganisms are selected so as to be capable of successfully
competing in the particular environment with the wild-type
microorganisms, provide for stable maintenance and expression of
the gene expressing the pesticidal protein, and desirably, provide
for improved protection of the pesticide from environmental
degradation and inactivation.
[0181] Such microorganisms include bacteria, algae, and fungi. Of
particular interest are microorganisms such as bacteria, e.g.,
Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas,
Streptomyces, Rhizobium, Rhodopseudomonas, Methylius,
Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,
Azotobacter, Leuconostoc, and Alcaligenes, fungi, particularly
yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,
Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular
interest are such phytosphere bacterial species as Pseudomonas
syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter
xylinum, Agrobacteria, Rhodopseudomonas spheroides, Xanthomonas
campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter
xyli and Azotobacter vinelandii and phytosphere yeast species such
as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,
Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces
rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S.
odorus, Kluyveromyces veronae, and Aureobasidium pollulans. Of
particular interest are the pigmented microorganisms.
[0182] A number of ways are available for introducing a gene
expressing the pesticidal protein into the microorganism host under
conditions that allow for stable maintenance and expression of the
gene. For example, expression cassettes can be constructed which
include the nucleotide constructs of interest operably linked with
the transcriptional and translational regulatory signals for
expression of the nucleotide constructs, and a nucleotide sequence
homologous with a sequence in the host organism, whereby
integration will occur, and/or a replication system that is
functional in the host, whereby integration or stable maintenance
will occur.
[0183] Transcriptional and translational regulatory signals
include, but are not limited to, promoters, transcriptional
initiation start sites, operators, activators, enhancers, other
regulatory elements, ribosomal binding sites, an initiation codon,
termination signals, and the like. See, for example, U.S. Pat. Nos.
5,039,523 and 4,853,331; EPO 0480762A2; Sambrook; Maniatis et al.
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) and the
references cited therein.
[0184] Suitable host cells, where the pesticidal protein-containing
cells will be treated to prolong the activity of the pesticidal
proteins in the cell when the treated cell is applied to the
environment of the target pest(s), may include either prokaryotes
or eukaryotes, normally being limited to those cells that do not
produce substances toxic to higher organisms, such as mammals.
However, organisms that produce substances toxic to higher
organisms could be used, where the toxin is unstable or the level
of application sufficiently low as to avoid any possibility of
toxicity to a mammalian host. As hosts, of particular interest will
be the prokaryotes and the lower eukaryotes, such as fungi.
Illustrative prokaryotes, both Gram-negative and gram-positive,
include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella,
Salmonella, and Proteus; Bacillaceae; Rhizobiaceae, such as
Rhizobium; Spirillaceae, such as photobacterium, Zymomonas,
Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum;
Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and
Acetobacter; Azotobacteraceae and Nitrobacteraceae. Among
eukaryotes are fungi, such as Phycomycetes and Ascomycetes, which
includes yeast, such as Saccharomyces and Schizosaccharomyces; and
Basidiomycetes yeast, such as Rhodotorula, Aureobasidium,
Sporobolomyces, and the like.
[0185] Characteristics of particular interest in selecting a host
cell for purposes of pesticidal protein production include ease of
introducing the pesticidal protein gene into the host, availability
of expression systems, efficiency of expression, stability of the
protein in the host, and the presence of auxiliary genetic
capabilities. Characteristics of interest for use as a pesticide
microcapsule include protective qualities for the pesticide, such
as thick cell walls, pigmentation, and intracellular packaging or
formation of inclusion bodies; leaf affinity; lack of mammalian
toxicity; attractiveness to pests for ingestion; ease of killing
and fixing without damage to the toxin; and the like. Other
considerations include ease of formulation and handling, economics,
storage stability, and the like.
[0186] Host organisms of particular interest include yeast, such as
Rhodotorula spp., Aureobasidium spp., Saccharomyces spp. (such as
S. cerevisiae), Sporobolomyces spp., phylloplane organisms such as
Pseudomonas spp. (such as P. aeruginosa, P. fluorescens), Erwinia
spp., and Flavobacterium spp., and other such organisms, including
Bt, E. coli, Bacillus subtilis, and the like.
[0187] Genes encoding the pesticidal proteins of the embodiments
can be introduced into microorganisms that multiply on plants
(epiphytes) to deliver pesticidal proteins to potential target
pests. Epiphytes, for example, can be gram-positive or
gram-negative bacteria.
[0188] Root-colonizing bacteria, for example, can be isolated from
the plant of interest by methods known in the art. Specifically, a
Bacillus cereus strain that colonizes roots can be isolated from
roots of a plant (see, for example, Handelsman et al. (1991) Appl.
Environ. Microbiol. 56:713-718). Genes encoding the pesticidal
proteins of the embodiments can be introduced into a
root-colonizing Bacillus cereus by standard methods known in the
art.
[0189] Genes encoding pesticidal proteins can be introduced, for
example, into the root-colonizing Bacillus by means of electro
transformation. Specifically, genes encoding the pesticidal
proteins can be cloned into a shuttle vector, for example, pHT3101
(Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218. The
shuttle vector pHT3101 containing the coding sequence for the
particular pesticidal protein gene can, for example, be transformed
into the root-colonizing Bacillus by means of electroporation
(Lerecius et al. (1989) FEMS Microbiol. Letts. 60: 211-218).
[0190] Expression systems can be designed so that pesticidal
proteins are secreted outside the cytoplasm of gram-negative
bacteria, such as E. coli, for example. Advantages of having
pesticidal proteins secreted are: (1) avoidance of potential
cytotoxic effects of the pesticidal protein expressed; and (2)
improvement in the efficiency of purification of the pesticidal
protein, including, but not limited to, increased efficiency in the
recovery and purification of the protein per volume cell broth and
decreased time and/or costs of recovery and purification per unit
protein.
[0191] Pesticidal proteins can be made to be secreted in E. coli,
for example, by fusing an appropriate E. coli signal peptide to the
amino-terminal end of the pesticidal protein. Signal peptides
recognized by E. coli can be found in proteins already known to be
secreted in E. coli, for example the OmpA protein (Ghrayeb et al.
(1984) EMBO J, 3:2437-2442). OmpA is a major protein of the E. coli
outer membrane, and thus its signal peptide is thought to be
efficient in the translocation process. Also, the OmpA signal
peptide does not need to be modified before processing as may be
the case for other signal peptides, for example lipoprotein signal
peptide (Duffaud et al. (1987) Meth. Enzymol. 153: 492).
[0192] Pesticidal proteins of the embodiments can be fermented in a
bacterial host and the resulting bacteria processed and used as a
microbial spray in the same manner that Bt strains have been used
as insecticidal sprays. In the case of a pesticidal protein(s) that
is secreted from Bacillus, the secretion signal is removed or
mutated using procedures known in the art. Such mutations and/or
deletions prevent secretion of the pesticidal protein(s) into the
growth medium during the fermentation process. The pesticidal
proteins are retained within the cell, and the cells are then
processed to yield the encapsulated pesticidal proteins. Any
suitable microorganism can be used for this purpose. Pseudomonas
has been used to express Bt toxins as encapsulated proteins and the
resulting cells processed and sprayed as an insecticide (Gaertner
et al. (1993), in: Advanced Engineered Pesticides, ed. Kim).
[0193] Alternatively, the pesticidal proteins are produced by
introducing a heterologous gene into a cellular host. Expression of
the heterologous gene results, directly or indirectly, in the
intracellular production and maintenance of the pesticide. These
cells are then treated under conditions that prolong the activity
of the toxin produced in the cell when the cell is applied to the
environment of target pest(s). The resulting product retains the
toxicity of the toxin. These naturally encapsulated pesticidal
proteins may then be formulated in accordance with conventional
techniques for application to the environment hosting a target
pest, e.g., soil, water, and foliage of plants. See, for example
EP0192319, and the references cited therein.
[0194] In the embodiments, a transformed microorganism (which
includes whole organisms, cells, spore(s), pesticidal protein(s),
pesticidal component(s), pest-impacting component(s), mutant(s),
living or dead cells and cell components, including mixtures of
living and dead cells and cell components, and including broken
cells and cell components) or an isolated pesticidal protein can be
formulated with an acceptable carrier into a pesticidal
composition(s) that is, for example, a suspension, a solution, an
emulsion, a dusting powder, a dispersible granule or pellet, a
wettable powder, and an emulsifiable concentrate, an aerosol or
spray, an impregnated granule, an adjuvant, a coatable paste, a
colloid, and also encapsulations in, for example, polymer
substances. Such formulated compositions may be prepared by such
conventional means as desiccation, lyophilization, homogenization,
extraction, filtration, centrifugation, sedimentation, or
concentration of a culture of cells comprising the polypeptide.
[0195] Such compositions disclosed above may be obtained by the
addition of a surface-active agent, an inert carrier, a
preservative, a humectant, a feeding stimulant, an attractant, an
encapsulating agent, a binder, an emulsifier, a dye, a UV
protectant, a buffer, a flow agent or fertilizers, micronutrient
donors, or other preparations that influence plant growth. One or
more agrochemicals including, but not limited to, herbicides,
insecticides, fungicides, bactericides, nematicides, molluscicides,
acaricides, plant growth regulators, harvest aids, and fertilizers,
can be combined with carriers, surfactants or adjuvants customarily
employed in the art of formulation or other components to
facilitate product handling and application for particular target
pests. Suitable carriers and adjuvants can be solid or liquid and
correspond to the substances ordinarily employed in formulation
technology, e.g., natural or regenerated mineral substances,
solvents, dispersants, wetting agents, tackifiers, binders, or
fertilizers. The active ingredients of the embodiments are normally
applied in the form of compositions and can be applied to the crop
area, plant, or seed to be treated. For example, the compositions
of the embodiments may be applied to grain in preparation for or
during storage in a grain bin or silo, etc. The compositions of the
embodiments may be applied simultaneously or in succession with
other compounds. Methods of applying an active ingredient of the
embodiments or an agrochemical composition of the embodiments that
contains at least one of the pesticidal proteins produced by the
bacterial strains of the embodiments include, but are not limited
to, foliar application, seed coating, and soil application. The
number of applications and the rate of application depend on the
intensity of infestation by the corresponding pest.
[0196] Suitable surface-active agents include, but are not limited
to, anionic compounds such as a carboxylate of, for example, a
metal; a carboxylate of a long chain fatty acid; an
N-acylsarcosinate; mono or di-esters of phosphoric acid with fatty
alcohol ethoxylates or salts of such esters; fatty alcohol sulfates
such as sodium dodecyl sulfate, sodium octadecyl sulfate or sodium
cetyl sulfate; ethoxylated fatty alcohol sulfates; ethoxylated
alkylphenol sulfates; lignin sulfonates; petroleum sulfonates;
alkyl aryl sulfonates such as alkyl-benzene sulfonates or lower
alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;
salts of sulfonated naphthalene-formaldehyde condensates; salts of
sulfonated phenol-formaldehyde condensates; more complex sulfonates
such as the amide sulfonates, e.g., the sulfonated condensation
product of oleic acid and N-methyl taurine; or the dialkyl
sulfosuccinates, e.g., the sodium sulfonate of dioctyl succinate.
Non-ionic agents include condensation products of fatty acid
esters, fatty alcohols, fatty acid amides or fatty-alkyl- or
alkenyl-substituted phenols with ethylene oxide, fatty esters of
polyhydric alcohol ethers, e.g., sorbitan fatty acid esters,
condensation products of such esters with ethylene oxide, e.g.,
polyoxyethylene sorbitar fatty acid esters, block copolymers of
ethylene oxide and propylene oxide, acetylenic glycols such as
2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic
glycols. Examples of a cationic surface-active agent include, for
instance, an aliphatic mono-, di-, or polyamine such as an acetate,
naphthenate or oleate; or oxygen-containing amine such as an amine
oxide of polyoxyethylene alkylamine; an amide-linked amine prepared
by the condensation of a carboxylic acid with a di- or polyamine;
or a quaternary ammonium salt.
[0197] Examples of inert materials include but are not limited to
inorganic minerals such as kaolin, phyllosilicates, carbonates,
sulfates, phosphates, or botanical materials such as cork, powdered
corncobs, peanut hulls, rice hulls, and walnut shells.
[0198] The compositions of the embodiments can be in a suitable
form for direct application or as a concentrate of primary
composition that requires dilution with a suitable quantity of
water or other diluent before application. The pesticidal
concentration will vary depending upon the nature of the particular
formulation, specifically, whether it is a concentrate or to be
used directly. The composition contains 1 to 98% of a solid or
liquid inert carrier, and 0 to 50% or 0.1 to 50% of a surfactant.
These compositions will be administered at the labeled rate for the
commercial product, for example, about 0.01 lb-5.0 lb. per acre
when in dry form and at about 0.01 pts.-10 pts. per acre when in
liquid form.
[0199] In a further embodiment, the compositions, as well as the
transformed microorganisms and pesticidal proteins of the
embodiments, can be treated prior to formulation to prolong the
pesticidal activity when applied to the environment of a target
pest as long as the pretreatment is not deleterious to the
pesticidal activity. Such treatment can be by chemical and/or
physical means as long as the treatment does not deleteriously
affect the properties of the composition(s). Examples of chemical
reagents include but are not limited to halogenating agents;
aldehydes such as formaldehyde and glutaraldehyde; anti-infectives,
such as zephiran chloride; alcohols, such as isopropanol and
ethanol; and histological fixatives, such as Bouin's fixative and
Helly's fixative (see, for example, Humason (1967) Animal Tissue
Techniques (W.H. Freeman and Co.).
[0200] In other embodiments, it may be advantageous to treat the
Cry toxin polypeptides with a protease, for example trypsin, to
activate the protein prior to application of a pesticidal protein
composition of the embodiments to the environment of the target
pest. Methods for the activation of protoxin by a serine protease
are well known in the art. See, for example, Cooksey (1968)
Biochem. J. 6:445-454 and Carroll and Ellar (1989) Biochem. J.
261:99-105, the teachings of which are herein incorporated by
reference. For example, a suitable activation protocol includes,
but is not limited to, combining a polypeptide to be activated, for
example a purified novel Cry polypeptide (e.g., having the amino
acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 8, and
trypsin at a 1/100 weight ratio of protein/trypsin in 20 nM
NaHCO.sub.3, pH 8 and digesting the sample at 36.degree. C. for 3
hours.
[0201] The compositions (including the transformed microorganisms
and pesticidal proteins of the embodiments) can be applied to the
environment of an insect pest by, for example, spraying, atomizing,
dusting, scattering, coating or pouring, introducing into or on the
soil, introducing into irrigation water, by seed treatment or
general application or dusting at the time when the pest has begun
to appear or before the appearance of pests as a protective
measure. For example, the pesticidal protein and/or transformed
microorganisms of the embodiments may be mixed with grain to
protect the grain during storage. It is generally important to
obtain good control of pests in the early stages of plant growth,
as this is the time when the plant can be most severely damaged.
The compositions of the embodiments can conveniently contain
another insecticide if this is thought necessary. In one
embodiment, the composition is applied directly to the soil, at a
time of planting, in granular form of a composition of a carrier
and dead cells of a Bacillus strain or transformed microorganism of
the embodiments. Another embodiment is a granular form of a
composition comprising an agrochemical such as, for example, an
herbicide, an insecticide, a fertilizer, an inert carrier, and dead
cells of a Bacillus strain or transformed microorganism of the
embodiments.
[0202] Those skilled in the art will recognize that not all
compounds are equally effective against all pests. Compounds of the
embodiments display activity against insect pests, which may
include economically important agronomic, forest, greenhouse,
nursery, ornamentals, food and fiber, public and animal health,
domestic and commercial structure, household, and stored product
pests. Insect pests include insects selected from the orders
Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga,
Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera,
Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly
Coleoptera and Lepidoptera.
[0203] Insects of the order Lepidoptera include, but are not
limited to, armyworms, cutworms, loopers, and heliothines in the
family Noctuidae: Agrotis ipsilon Hufnagel (black cutworm); A.
orthogonia Morrison (western cutworm); A. segetum Denis &
Schiffermuller (turnip moth); A. subterranea Fabricius (granulate
cutworm); Alabama argillacea Hubner (cotton leaf worm); Anticarsia
gemmatalis Hubner (velvetbean caterpillar); Athetis mindara Barnes
and McDunnough (rough skinned cutworm); Earias insulana Boisduval
(spiny bollworm); E. vittella Fabricius (spotted bollworm); Egira
(Xylomyges) curiails Grote (citrus cutworm); Euxoa messoria Harris
(darksided cutworm); Helicoverpa armigera Hubner (American
bollworm); H. zea Boddie (corn earworm or cotton bollworm);
Heliothis virescens Fabricius (tobacco budworm); Hypena scabra
Fabricius (green cloverworm); Mamestra configurata Walker (bertha
armyworm); M. brassicae Linnaeus (cabbage moth); Melanchra picta
Harris (zebra caterpillar); Pseudaletia unipuncta Haworth
(armyworm); Pseudoplusia includens Walker (soybean looper); Richia
albicosta Smith (Western bean cutworm); Spodoptera frugiperda JE
Smith (fall armyworm); S. exigua Hubner (beet armyworm); S. litura
Fabricius (tobacco cutworm, cluster caterpillar); Trichoplusia ni
Hubner (cabbage looper); borers, casebearers, webworms, coneworms,
and skeletonizers from the families Pyralidae and Crambidae such as
Achroia grisella Fabricius (lesser wax moth); Amyelois transitella
Walker (naval orangeworm); Anagasta kuehniella Zeller
(Mediterranean flour moth); Cadra cautella Walker (almond moth);
Chilo partellus Swinhoe (spotted stalk borer); C. suppressalis
Walker (striped stem/rice borer); C. terrenellus Pagenstecher
(sugarcane stemp borer); Corcyra cephalonica Stainton (rice moth);
Crambus caliginosellus Clemens (corn root webworm); C. teterrellus
Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice
leaf roller); Desmia funeralis Hubner (grape leaffolder); Diaphania
hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm);
Diatraea grandiosella Dyar (southwestern corn borer), D.
saccharalis Fabricius (surgarcane borer); Elasmopalpus lignosellus
Zeller (lesser cornstalk borer); Eoreuma loftini Dyar (Mexican rice
borer); Ephestia elutella Hubner (tobacco (cacao) moth); Galleria
mellonella Linnaeus (greater wax moth); Hedylepta accepta Butler
(sugarcane leafroller); Herpetogramma licarsisalis Walker (sod
webworm); Homoeosoma electellum Hulst (sunflower moth); Loxostege
sticticalis Linnaeus (beet webworm); Maruca testulalis Geyer (bean
pod borer); Orthaga thyrisalis Walker (tea tree web moth); Ostrinia
nubilalis Hubner (European corn borer); Plodia interpunctella
Hubner (Indian meal moth); Scirpophaga incertulas Walker (yellow
stem borer); Udea rubigalis Guenee (celery leaftier); and
leafrollers, budworms, seed worms, and fruit worms in the family
Tortricidae Acleris gloverana Walsingham (Western blackheaded
budworm); A. variana Fernald (Eastern blackheaded budworm);
Adoxophyes orana Fischer von Rosslerstamm (summer fruit tortrix
moth); Archips spp. including A. argyrospila Walker (fruit tree
leaf roller) and A. rosana Linnaeus (European leaf roller);
Argyrotaenia spp.; Bonagota salubricola Meyrick (Brazilian apple
leafroller); Choristoneura spp.; Cochylis hospes Walsingham (banded
sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.
pomonella Linnaeus (codling moth); Endopiza viteana Clemens (grape
berry moth); Eupoecilia ambiguella Hubner (vine moth); Grapholita
molesta Busck (oriental fruit moth); Lobesia botrana Denis &
Schiffermuller (European grape vine moth); Platynota flavedana
Clemens (variegated leafroller); P. stultana Walsingham (omnivorous
leafroller); Spilonota ocellana Denis & Schiffermuller
(eyespotted bud moth); and Suleima helianthana Riley (sunflower bud
moth).
[0204] Selected other agronomic pests in the order Lepidoptera
include, but are not limited to, Alsophila pometaria Harris (fall
cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota
senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi
Guerin-Meneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus
(Silkworm); Bucculatrix thurberiella Busck (cotton leaf
perforator); Collas eurytheme Boisduval (alfalfa caterpillar);
Datana integerrima Grote & Robinson (walnut caterpillar);
Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos
subsignaria Hubner (elm spanworm); Erannis tiliaria Harris (linden
looper); Erechthias flavistriata Walsingham (sugarcane bud moth);
Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina
americana Guerin-Meneville (grapeleaf skeletonizer); Heliothis
subflexa Guenee; Hemileuca oliviae Cockrell (range caterpillar);
Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella
Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst
(Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western
hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria
dispar Linnaeus (gypsy moth); Malacosoma spp.; Manduca
quinquemaculata Haworth (five spotted hawk moth, tomato hornworm);
M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera
brumata Linnaeus (winter moth); Orgyia spp.; Paleacrita vernata
Peck (spring cankerworm); Papilio cresphontes Cramer (giant
swallowtail, orange dog); Phryganidia californica Packard
(California oakworm); Phyllocnistis citrella Stainton (citrus
leafminer); Phyllonorycter blancardella Fabricius (spotted
tentiform leafminer); Pieris brassicae Linnaeus (large white
butterfly); P. rapae Linnaeus (small white butterfly); P. napi
Linnaeus (green veined white butterfly); Platyptilia carduidactyla
Riley (artichoke plume moth); Plutella xylostella Linnaeus
(diamondback moth); Pectinophora gossypiella Saunders (pink
bollworm); Pontia protodice Boisduval & Leconte (Southern
cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper);
Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga
cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa
Schiffermuller (pine processionary caterpillar); Tineola
bisselliella Hummel (webbing clothesmoth); Tuta absolute Meyrick
(tomato leafminer) and Yponomeuta padella Linnaeus (ermine
moth).
[0205] Of interest are larvae and adults of the order Coleoptera
including weevils from the families Anthribidae, Bruchidae, and
Curculionidae including, but not limited to: Anthonomus grandis
Boheman (boll weevil); Cylindrocopturus adspersus LeConte
(sunflower stem weevil); Diaprepes abbreviatus Linnaeus (Diaprepes
root weevil); Hypera punctata Fabricius (clover leaf weevil);
Lissorhoptrus oryzophilus Kuschel (rice water weevil); Metamasius
hemipterus hemipterus Linnaeus (West Indian cane weevil); M.
hemipterus sericeus Olivier (silky cane weevil); Sitophilus
granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice
weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S.
sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis
Chittenden (maize billbug); Rhabdoscelus obscurus Boisduval (New
Guinea sugarcane weevil); flea beetles, cucumber beetles,
rootworms, leaf beetles, potato beetles, and leafminers in the
family Chrysomelidae including, but not limited to: Chaetocnema
ectypa Horn (desert corn flea beetle); C. pulicaria Melsheimer
(corn flea beetle); Colaspis brunnea Fabricius (grape colaspis);
Diabrotica barberi Smith & Lawrence (northern corn rootworm);
D. undecimpunctata howardi Barber (southern corn rootworm); D.
virgifera virgifera LeConte (western corn rootworm); Leptinotarsa
decemlineata Say (Colorado potato beetle); Oulema melanopus
Linnaeus (cereal leaf beetle); Phyllotreta cruciferae Goeze (corn
flea beetle); Zygogramma exclamationis Fabricius (sunflower
beetle); beetles from the family Coccinellidae including, but not
limited to: Epilachna varivestis Mulsant (Mexican bean beetle);
chafers and other beetles from the family Scarabaeidae including,
but not limited to: Antitrogus parvulus Britton (Childers cane
grub); Cyclocephala borealis Arrow (northern masked chafer, white
grub); C. immaculate Olivier (southern masked chafer, white grub);
Dermolepida albohirtum Waterhouse (Greyback cane beetle); Euetheola
humilis rugiceps LeConte (sugarcane beetle); Lepidiota frenchi
Blackburn (French's cane grub); Tomarus gibbosus De Geer (carrot
beetle); T. subtropicus Blatchley (sugarcane grub); Phyllophaga
crinita Burmeister (white grub); P. latifrons LeConte (June
beetle); Popillia japonica Newman (Japanese beetle); Rhizotrogus
majalis Razoumowsky (European chafer); carpet beetles from the
family Dermestidae; wireworms from the family Elateridae, Eleodes
spp., Melanotus spp. including M. communis Gyllenhal (wireworm);
Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.;
Aeolus spp.; bark beetles from the family Scolytidae; beetles from
the family Tenebrionidae; beetles from the family Cerambycidae such
as, but not limited to, Migdolus fryanus Westwood (longhorn
beetle); and beetles from the Buprestidae family including, but not
limited to, Aphanisticus cochinchinae seminulum Obenberger
(leaf-mining buprestid beetle).
[0206] Adults and immatures of the order Diptera are of interest,
including leafminers Agromyza parvicornis Loew (corn blotch
leafminer); midges including, but not limited to: Contarinia
sorghicola Coquillett (sorghum midge); Mayetiola destructor Say
(Hessian fly); Neolasioptera murtfeldtiana Felt, (sunflower seed
midge); Sitodiplosis mosellana Gehin (wheat midge); fruit flies
(Tephritidae), Oscinella frit Linnaeus (frit flies); maggots
including, but not limited to: Delia spp. including Delia platura
Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly);
Fannia canicularis Linnaeus, F. femoralis Stein (lesser house
flies); Meromyza americana Fitch (wheat stem maggot); Musca
domestica Linnaeus (house flies); Stomoxys calcitrans Linnaeus
(stable flies)); face flies, horn flies, blow flies, Chrysomya
spp.; Phormia spp.; and other muscoid fly pests, horse flies
Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle
grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus
Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.;
Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium
spp.; biting midges, sand flies, sciarids, and other
Nematocera.
[0207] Included as insects of interest are those of the order
Hemiptera such as, but not limited to, the following families:
Adelgidae, Aleyrodidae, Aphididae, Asterolecaniidae, Cercopidae,
Cicadellidae, Cicadidae, Cixiidae, Coccidae, Coreidae,
Dactylopiidae, Delphacidae, Diaspididae, Eriococcidae, Flatidae,
Fulgoridae, lssidae, Lygaeidae, Margarodidae, Membracidae, Miridae,
Ortheziidae, Pentatomidae, Phoenicococcidae, Phylloxeridae,
Pseudococcidae, Psyllidae, Pyrrhocoridae and Tingidae.
[0208] Agronomically important members from the order Hemiptera
include, but are not limited to: Acrosternum hilare Say (green
stink bug); Acyrthisiphon pisum Harris (pea aphid); Adelges spp.
(adelgids); Adelphocoris rapidus Say (rapid plant bug); Anasa
tristis De Geer (squash bug); Aphis craccivora Koch (cowpea aphid);
A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton
aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A.
pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid);
Aulacaspis tegalensis Zehntner (sugarcane scale); Aulacorthum
solani Kaltenbach (foxglove aphid); Bemisia tabaci Gennadius
(tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows
& Perring (silverleaf whitefly); Blissus leucopterus
leucopterus Say (chinch bug); Blostomatidae spp.; Brevicoryne
brassicae Linnaeus (cabbage aphid); Cacopsylla pyricola Foerster
(pear psylla); Calocoris norvegicus Gmelin (potato capsid bug);
Chaetosiphon fragaefolii Cockerell (strawberry aphid); Cimicidae
spp.; Coreidae spp.; Corythuca gossypii Fabricius (cotton lace
bug); Cyrtopeltis modesta Distant (tomato bug); C. notatus Distant
(suckfly); Deois flavopicta Stal (spittlebug); Dialeurodes citri
Ashmead (citrus whitefly); Diaphnocoris chlorionis Say (honeylocust
plant bug); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat
aphid); Duplachionaspis divergens Green (armored scale); Dysaphis
plantaginea Paaserini (rosy apple aphid); Dysdercus suturellus
Herrich-Schaffer (cotton stainer); Dysmicoccus boninsis Kuwana
(gray sugarcane mealybug); Empoasca fabae Harris (potato
leafhopper); Eriosoma lanigerum Hausmann (woolly apple aphid);
Erythroneoura spp. (grape leafhoppers); Eumetopina flavipes Muir
(Island sugarcane planthopper); Eurygaster spp.; Euschistus servus
Say (brown stink bug); E. variolarius Palisot de Beauvois
(one-spotted stink bug); Graptostethus spp. (complex of seed bugs);
and Hyalopterus pruni Geoffroy (mealy plum aphid); Icerya purchasi
Maskell (cottony cushion scale); Labopidicola affii Knight (onion
plant bug); Laodelphax striatellus Fallen (smaller brown
planthopper); Leptoglossus corculus Say (leaf-footed pine seed
bug); Leptodictya tabida Herrich-Schaeffer (sugarcane lace bug);
Lipaphis erysimi Kaltenbach (turnip aphid); Lygocoris pabulinus
Linnaeus (common green capsid); Lygus lineolaris Palisot de
Beauvois (tarnished plant bug); L. Hesperus Knight (Western
tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L.
rugulipennis Poppius (European tarnished plant bug); Macrosiphum
euphorbiae Thomas (potato aphid); Macrosteles quadrilineatus Forbes
(aster leafhopper); Magicicada septendecim Linnaeus (periodical
cicada); Mahanarva fimbriolata Stal (sugarcane spittlebug);
Melanaphis sacchari Zehntner (sugarcane aphid); Melanaspis
glomerata Green (black scale); Metopolophium dirhodum Walker (rose
grain aphid); Myzus persicae Sulzer (peach-potato aphid, green
peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);
Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal
(rice leafhopper); Nezara viridula Linnaeus (southern green stink
bug); Nilaparvata lugens Stal (brown planthopper); Nysius ericae
Schilling (false chinch bug); Nysius raphanus Howard (false chinch
bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus
fasciatus Dallas (large milkweed bug); Orthops campestris Linnaeus;
Pemphigus spp. (root aphids and gall aphids); Peregrinus maidis
Ashmead (corn planthopper); Perkinsiella saccharicida Kirkaldy
(sugarcane delphacid); Phylloxera devastatrix Pergande (pecan
phylloxera); Planococcus citri Risso (citrus mealybug); Plesiocoris
rugicoffis Fallen (apple capsid); Poecilocapsus lineatus Fabricius
(four-lined plant bug); Pseudatomoscelis seriatus Reuter (cotton
fleahopper); Pseudococcus spp. (other mealybug complex); Pulvinaria
elongata Newstead (cottony grass scale); Pyrilla perpusilla Walker
(sugarcane leafhopper); Pyrrhocoridae spp.; Quadraspidiotus
perniciosus Comstock (San Jose scale); Reduviidae spp.;
Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus
(bird cherry-oat aphid); Saccharicoccus sacchari Cockerell (pink
sugarcane mealybug); Schizaphis graminum Rondani (greenbug); Sipha
flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius
(English grain aphid); Sogatella furcifera Horvath (white-backed
planthopper); Sogatodes oryzicola Muir (rice delphacid);
Spanagonicus albofasciatus Reuter (whitemarked fleahopper);
Therioaphis maculata Buckton (spotted alfalfa aphid); Tinidae spp.;
Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and
T. citricida Kirkaldy (brown citrus aphid); Trialeurodes
abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood
(greenhouse whitefly); Trioza diospyri Ashmead (persimmon psylla);
and Typhlocyba pomaria McAtee (white apple leafhopper).
[0209] Also included are adults and larvae of the order Acari
(mites) such as Aceria tosichella Keifer (wheat curl mite);
Panonychus ulmi Koch (European red mite); Petrobia latens Muller
(brown wheat mite); Steneotarsonemus bancrofti Michael (sugarcane
stalk mite); spider mites and red mites in the family
Tetranychidae, Oligonychus grypus Baker & Pritchard, O. indicus
Hirst (sugarcane leaf mite), O. pratensis Banks (Banks grass mite),
O. stickneyi McGregor (sugarcane spider mite); Tetranychus urticae
Koch (two spotted spider mite); T. mcdanieli McGregor (McDaniel
mite); T. cinnabarinus Boisduval (carmine spider mite); T.
turkestani Ugarov & Nikolski (strawberry spider mite), flat
mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor
(citrus flat mite); rust and bud mites in the family Eriophyidae
and other foliar feeding mites and mites important in human and
animal health, i.e. dust mites in the family Epidermoptidae,
follicle mites in the family Demodicidae, grain mites in the family
Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say
(deer tick); I. holocyclus Neumann (Australian paralysis tick);
Dermacentor variabilis Say (American dog tick); Amblyomma
americanum Linnaeus (lone star tick); and scab and itch mites in
the families Psoroptidae, Pyemotidae, and Sarcoptidae.
[0210] Insect pests of the order Thysanura are of interest, such as
Lepisma saccharina Linnaeus (silverfish); Thermobia domestica
Packard (firebrat).
[0211] Additional arthropod pests covered include: spiders in the
order Araneae such as Loxosceles reclusa Gertsch & Mulaik
(brown recluse spider); and the Latrodectus mactans Fabricius
(black widow spider); and centipedes in the order Scutigeromorpha
such as Scutigera coleoptrata Linnaeus (house centipede). In
addition, insect pests of the order Isoptera are of interest,
including those of the termitidae family, such as, but not limited
to, Cylindrotermes nordenskioeldi Holmgren and Pseudacanthotermes
militaris Hagen (sugarcane termite). Insects of the order
Thysanoptera are also of interest, including but not limited to
thrips, such as Stenchaetothrips minutus van Deventer (sugarcane
thrips).
[0212] Insect pests may be tested for pesticidal activity of
compositions of the embodiments in early developmental stages,
e.g., as larvae or other immature forms. The insects may be reared
in total darkness at from about 20.degree. C. to about 30.degree.
C. and from about 30% to about 70% relative humidity. Bioassays may
be performed as described in Czapla and Lang (1990) J. Econ.
Entomol. 83(6): 2480-2485. Methods of rearing insect larvae and
performing bioassays are well known to one of ordinary skill in the
art.
[0213] A wide variety of bioassay techniques are known to one
skilled in the art. General procedures include addition of the
experimental compound or organism to the diet source in an enclosed
container. Pesticidal activity can be measured by, but is not
limited to, changes in mortality, weight loss, attraction,
repellency and other behavioral and physical changes after feeding
and exposure for an appropriate length of time. Bioassays described
herein can be used with any feeding insect pest in the larval or
adult stage.
[0214] The following examples are presented by way of illustration,
not by way of limitation.
EXPERIMENTALS
Example 1--Generation of Cry1B Variants with Improved Spectrum of
Insecticidal Activity
[0215] The Cry1Bd insecticidal protein having an amino acid of SEQ
ID NO: 1 (U.S. Pat. No. 8,692,065) has high insecticidal activity
(ILC50=1 ppm) against European corn borer (Ostrinia nubilalis)
larvae but low insecticidal activity (ILC50>1000 ppm and
.about.400 ppm respectively) against corn earworm (Helicoverpa zea)
and fall armyworm (Spodoptera frugiperda). The Cry1B insecticidal
protein, referred to as MP258 (Serial No. PCT/US14/49923) having an
amino acid of SEQ ID NO: 47 has high insecticidal activity (ILC50=4
ppm) against European corn borer (Ostrinia nubilalis) larvae but
lower insecticidal activity (ILC50 24 ppm and 62 ppm respectively)
against corn earworm (Helicoverpa zea) and fall armyworm
(Spodoptera frugiperda). A series of variant Cry1B polypeptides
derived from Cry1Bd (SEQ ID NO: 1) and MP258 were designed to
improve the insecticidal activity against corn earworm (CEW) and/or
fall armyworm (FAW) compared to Cry1Bd (SEQ ID NO: 1) and/or MP258
(SEQ ID NO: 47) while maintaining the ECB insecticidal activity.
Variant Cry1B polypeptides having improved insecticidal activity
that were generated include those indicated in Table 1. The
insecticidal activity of the Cry1B variants was determined as
described in Example 4 and the insecticidal activity results are
shown in Table 3. An amino acid sequence alignment of the variant
Cry1B polypeptides is shown in FIG. 1.
TABLE-US-00001 TABLE 1 Clone ID Polypeptide Polynucleotide Cry1Bd
SEQ ID NO: 1 SEQ ID NO: 2 IP1B-B1 SEQ ID NO: 3 SEQ ID NO: 4
IP1B-B21 SEQ ID NO: 5 SEQ ID NO: 6 IP1B-B22 SEQ ID NO: 7 SEQ ID NO:
8 IP1B-B23 SEQ ID NO: 9 SEQ ID NO: 10 IP1B-B24 SEQ ID NO: 11 SEQ ID
NO: 12 IP1B-B25 SEQ ID NO: 13 SEQ ID NO: 14 IP1B-B26 SEQ ID NO: 15
SEQ ID NO: 16 IP1B-B27 SEQ ID NO: 17 SEQ ID NO: 18 IP1B-B28 SEQ ID
NO: 19 SEQ ID NO: 20 IP1B-B29 SEQ ID NO: 21 SEQ ID NO: 22 IP1B-B31
SEQ ID NO: 23 SEQ ID NO: 24 IP1B-B32 SEQ ID NO: 25 SEQ ID NO: 26
IP1B-B33 SEQ ID NO: 27 SEQ ID NO: 28 IP1B-B34 SEQ ID NO: 29 SEQ ID
NO: 30 IP1B-B40 SEQ ID NO: 31 SEQ ID NO: 32 IP1B-B41 SEQ ID NO: 33
SEQ ID NO: 34 IP1B-B42 SEQ ID NO: 35 SEQ ID NO: 36 IP1B-B43 SEQ ID
NO: 37 SEQ ID NO: 38 IP1B-B44 SEQ ID NO: 39 SEQ ID NO: 40 IP1B-B45
SEQ ID NO: 41 SEQ ID NO: 42 IP1B-B46 SEQ ID NO: 43 SEQ ID NO: 44
IP1B-B47 SEQ ID NO: 45 SEQ ID NO: 46 MP258 SEQ ID NO: 47 SEQ ID NO:
48 GS060 SEQ ID NO: 49 SEQ ID NO: 50
[0216] The percent amino acid sequence identity of the Cry1B
variant polypeptides calculated using the Needleman-Wunsch
algorithm, as implemented in the Needle program (EMBOSS tool
suite), are shown as a matrix table in Table 2a-2b. The void part
of the matrix table is not shown.
TABLE-US-00002 TABLE 2a IP1B- IP1B- IP1B- IP1B- IP1B- IP1B- IP1B-
IP1B- IP1B- IP1B- GS060 B1 B21 B22 B23 B24 B25 B26 B27 B28 B29
Cry1Bd 65.6 95.4 84.3 82.6 82.5 84.3 84.3 84.2 83.7 83.7 83.7 GS060
-- 67.0 60.1 60.2 60.1 60.1 60.2 60.1 60.0 59.9 60.1 IP1B-B1 -- --
83.4 82.6 84.5 83.4 83.4 83.2 82.9 82.9 82.9 IP1B-B21 -- -- -- 95.4
96.9 99.7 99.7 99.5 99.1 99.1 99.1 IP1B-B22 -- -- -- -- 95.4 95.1
95.1 95.0 94.5 94.8 94.8 IP1B-B23 -- -- -- -- -- 96.6 96.6 96.5
96.0 96.0 96.0 IP1B-B24 -- -- -- -- -- -- 99.4 99.2 98.8 98.8 98.8
IP1B-B25 -- -- -- -- -- -- -- 99.8 99.4 99.4 99.4 IP1B-B26 -- -- --
-- -- -- -- -- 99.5 99.2 99.2 IP1B-B27 -- -- -- -- -- -- -- -- --
99.4 99.4 IP1B-B28 -- -- -- -- -- -- -- -- -- -- 99.8
TABLE-US-00003 TABLE 2b IP1B- IP1B- IP1B- IP1B- IP1B- IP1B- IP1B-
IP1B- IP1B- IP1B- IP1B- IP1B- B31 B32 B33 B34 B40 B41 B42 B43 B44
B45 B46 B47 MP258 Cry1Bd 80.4 80.4 81.0 82.0 83.7 83.9 83.9 83.9
83.9 83.9 83.9 83.9 82.3 GS060 66.6 66.9 66.3 65.5 59.8 59.9 60.1
60.1 60.1 60.1 59.9 59.9 59.9 IP1B-B1 83.6 83.0 82.7 81.6 82.8 82.9
83.1 83.1 83.1 83.1 83.1 83.1 80.9 IP1B-B21 71.6 71.5 71.8 71.8
99.1 99.1 99.2 99.2 99.2 99.2 99.2 99.2 96.9 IP1B-B22 70.7 70.4
70.7 71.0 94.7 94.7 94.7 94.7 94.7 94.7 94.8 94.8 97.6 IP1B-B23
72.5 72.3 72.6 72.3 96.0 96.0 96.2 96.2 96.2 96.2 96.2 96.2 96.0
IP1B-B24 71.6 71.5 71.8 71.8 98.8 98.9 98.9 98.9 98.9 98.9 98.9
98.9 96.6 IP1B-B25 71.8 71.6 71.9 71.9 99.4 99.4 99.5 99.5 99.5
99.5 99.5 99.5 96.6 IP1B-B26 71.6 71.5 71.8 71.8 99.5 99.2 99.4
99.4 99.4 99.4 99.4 99.4 96.5 IP1B-B27 71.3 71.2 71.5 71.3 99.2
98.9 99.7 99.5 99.5 99.5 99.2 99.2 96.0 IP1B-B28 71.3 71.2 71.5
71.3 99.1 99.1 99.4 99.2 99.2 99.2 99.5 99.5 96.3 IP1B-B29 71.3
71.2 71.5 71.3 99.1 99.1 99.4 99.2 99.2 99.2 99.4 99.4 96.3
IP1B-B31 -- 99.4 99.1 98.0 71.3 71.6 71.5 71.5 71.5 71.5 71.5 71.5
69.2 IP1B-B32 -- -- 99.2 98.0 71.2 71.5 71.3 71.3 71.3 71.3 71.3
71.3 69.1 IP1B-B33 -- -- -- 98.0 71.5 71.8 71.6 71.6 71.6 71.6 71.6
71.6 69.4 IP1B-B34 -- -- -- -- 71.5 71.8 71.5 71.5 71.5 71.5 71.5
71.5 69.7 IP1B-B40 -- -- -- -- -- 99.7 99.1 99.1 99.1 99.2 99.2
99.4 96.2 IP1B-B41 -- -- -- -- -- -- 99.1 99.1 99.1 99.2 99.2 99.4
96.2 IP1B-B42 -- -- -- -- -- -- -- 99.8 99.8 99.7 99.5 99.4 96.2
IP1B-B43 -- -- -- -- -- -- -- -- 99.8 99.8 99.5 99.5 96.2 IP1B-B44
-- -- -- -- -- -- -- -- -- 99.7 99.7 99.4 96.2 IP1B-B45 -- -- -- --
-- -- -- -- -- -- 99.4 99.7 96.2 IP1B-B46 -- -- -- -- -- -- -- --
-- -- -- 99.7 96.3 IP1B-B47 -- -- -- -- -- -- -- -- -- -- -- --
96.3
Example 2--Saturation Mutagenesis at Selected Positions of MP258
and IP-1B Variant Cry1B Polypeptides
[0217] The polynucleotides of SEQ ID NO: 48, SEQ ID NO: 6, SEQ ID
NO: 14, and SEQ ID NO: 42 encoding MP258, IP1B-B21, IP1B-B25 and
IP1B-B45 (SEQ ID NO: 47, SEQ ID NO: 5, SEQ ID NO: 13, and SEQ ID
NO: 41 respectively) were used as the templates for saturation
mutagenesis at selected amino acid positions. A reverse mutagenesis
primer and a complementary forward mutagenesis primer were designed
to create the desired amino acid substitution(s) at the site(s) of
interest. Typically the mutagenesis primer was between 30 to 45
bases in length with two or more bases, usually 10 to 15, on both
sides of the site of interest. In order to make saturation
mutagenesis, degenerated primers that cover all possible amino acid
residues were used. The mutagenic reactions were carried out using
Agilent's QuikChange.TM. Lightening Site-Directed Mutagenesis kit.
Materials provided in the kit are QuikChange.TM. Lightening Enzyme,
10.times. QuikChange.TM. Lightning Buffer, dNTP mix,
QuikSolution.TM. reagent and Don restriction enzyme according to
the manufactures directions.
[0218] PCR amplifications were typically carried out with
Expand.TM. High Fidelity PCR system (Roche, Switzerland) in 50 ul
containing 50-100 ng templates, 0.4-2 .mu.M primer pair, 200 .mu.M
dNTPs and 2 Units of DNA polymerase. The mutagenesis reaction was
initiated by pre-heating the reaction mixture to 94.degree. C. for
3 min, followed by 16 cycles of the following cycling program:
94.degree. C. for 1 min, 52.degree. C. for 1 min and 68.degree. C.
for 8, 12, 16 or 24 min according to the length of template. The
mutagenesis reaction was completed by incubation at 68.degree. C.
for 1 h. The PCR-amplification products were evaluated by agarose
gel electrophoresis. The PCR products were purified by QIAquick.TM.
PCR purification kit (Qiagen, Germany) and further treated with the
restriction enzyme Dpnl. An aliquot of 1 .mu.l of the PCR product
was typically transformed into BL21(DE3) cells and inoculated on
Luria-Bertani (LB) plate containing 100 .mu.g/ml ampicillin. About
48 or more colonies for saturation mutagenesis were selected and
plasmid DNA was isolated for sequencing. Two step sequencing was
used, first for specific mutation site(s) with one sequencing
primer followed by full length sequence confirmation with multiple
sequencing primers. After all 19 amino acid mutations were
confirmed by sequencing, those mutant genes were advanced for
expression and protein purification.
[0219] In the case of mutations made to cover the entire IP1B-B25
Domain III spanning from T495 to E655, 48 mutant clones were picked
from each site and screened for the CEW activity, as described in
Example 4. In order to sequence those mutant clones to determine
mutated amino acids, among 151 amino acid residues subjected to
mutagenesis, 103 sites were sequenced based on the number of
up-mutations and down-mutations. Those sites containing mutants
showing no significant activity changes were not sequenced.
Example 3--Purification of Variant cry1B Insecticidal Proteins
[0220] Variant cry1B insecticidal protein genes were expressed in a
modified pMAL vector (Cat# E8000S from New England Biolabs) as a
fusion with MBP (maltose binding protein). The pMAL vector was
modified to attach a 6.times.His tag to the N-terminal end of MBP
after methionine at position 1. The plasmid containing the
insecticidal protein gene was cloned in E. coli BL21 (DE3). The
BL21 cells were grown in MagicMedia.TM. (Life Technologies) in
either 96 deep well plates or flasks in a shaker running at 250 rpm
at 37.degree. C. for 8 hrs followed by 16.degree. C. for 64 hrs.
During the 16.degree. C. incubation, the MBP-toxin fusion protein
was accumulated in the BL21 cell as a soluble protein.
[0221] In order to purify the fusion protein, the E. coli cells
were harvested by centrifugation and treated in a lysozyme solution
consisting of 2 mg/ml lysozyme in 50 ml sodium phosphate buffer at
pH8 containing 300 mM NaCl, 2 U/ml endonuclease (Epicentre) and 5
mM MaCl2 for 3 hrs at 37.degree. C. with gentle shaking. The
lysozyme treated E. coli cells were then disrupted with 1% Triton
X100 and clear lysate containing the IP-1B proteins were prepared
by centrifugation at 4000 rpm, 30 min (96 well plates) or 9000 rpm
(flask produced samples). His tagged MBP-toxin proteins were
purified from the clear lysate by affinity chromatography using
NiNTA agarose from Qiagen.TM. following the manufacturer's standard
procedure. For those clear lysate samples made in 96 well plates,
Pall Corporation.TM. (25 Harbor Park Drive Port Washington, N.Y.
11050) 96 deep well filter plates were used as affinity
chromatography columns. The purified toxin proteins eluted from
NiNTA agarose was passed through Sephadex G25 to change the
phosphate buffer to 25 mM HEPES-NaOH, pH8 and used in insect
bioassay for determining the insecticidal. MBP was digested with
1/100 (w/w) Factor Xa (New England Biolabs) at 25.degree. C. for
overnight and removed from the IP-1B proteins by Superdex 200
column chromatography utilizing the size difference and a weak
affinity of MBP to Superdex.
[0222] Protein concentrations were determined by capillary
electrophoresis with the LabChip.TM. GXII device (Caliper
LifeSciences). The protein analysis was repeated at least 3 times
until the final concentrations were considered to be reliable
within the predetermined deviation, less than 10%.
Example 4--Determination of the Insecticidal Activity of Variant
IP-1B Proteins
[0223] The activity of Cry1B polypeptide variants against major
corn pests, European Corn Borer (ECB, Ostrinia nubilalis), Corn
Earworm (ECW, Helicoverpa zea) and Fall Armyworm (FAW, Spodoptera
frugiperda), was determined by feeding assay as described by Cong,
R., et al. Proceedings of the 4th Pacific Rim Conferences on
Biotechnology of Bacillus thuringiensis and its environmental
impact, pp. 118-123, ed. by R. J. Akhurst, C. E. Beard and P.
Hughes, published in 2002, Canberra, Australia. Briefly, the assays
were conducted on an artificial diet containing the insecticidal
proteins. The insecticidal proteins were prepared as described in
Example 1, and 10 .mu.L of protein samples were mixed with 40 .mu.L
of molten (40-50.degree. C.) artificial insect diet prepared based
on Southland Premix formulated for Lepidopteran insects (Southland
Products, Lake Village, Ark.) with low temperature melting agarose.
The diet-insecticidal protein mixture was placed in each well of a
96 well micro-titer plate. One or more neonate insect larvae were
placed in each well to feed for 4 days for CEW and FAW and 5 days
for ECB at 28.degree. C.
[0224] Alternatively, insect eggs or larvae were sorted by Large
Particle Flow Cytometry using COPAS.TM. (Complex Object Parametric
Analyzer and Sorter) obtained from Union Biometrica (Holliston,
Mass.) to place one egg or larva per well in a 96-well micro-titer
plate that contains solidified artificial insect diet. When eggs
were used to place in the assay plates, only those wells containing
hatched larvae after 16 hours were used for assay data collection.
Usually 90 to 95% hatch rates were obtained due to efficient COPAS
sorting. After certain feeding periods, the response of insects
towards the proteins was scored using a 0-3 numerical scoring
system based on the size and mortality of the larvae in each well.
If no response (or normal growth) was seen, a score of 0 was given.
When the growth was slightly retarded, a score of 1 was given. A
score of 2 meant that the larvae were severely retarded in growth
(close to neonate size). A score of 3 meant death to all the larvae
in the well. The percent response (Response) for each treatment was
calculated by dividing the total score, a sum of scores from
replicating wells for each treatment by the total highest possible
scores. For example, if one treatment (one sample, one dose) had 6
replicating wells, the total highest possible score would be
3.times.6=18.
[0225] In order to identify variant Cry1B polypeptides that have
increased levels of the activity toward those corn pests,
significantly higher than the activity reference such as the wild
type, non-mutated reference protein (e.g. MP258 SEQ ID NO: 47).
Variant polypeptides at certain concentrations were assayed along
with 4 doses of the reference protein within one 96-well assay
plate. The concentrations of the insecticidal proteins were within
the 4 doses of the reference protein concentrations, preferably
around the middle point of the 4 dose concentrations. Each sample
plate contained the reference protein in a significant number of
wells such as 16 wells in 4 separate doses. Also in each plate, up
to 80 mutants proteins for activity comparison with the reference
protein were included. From a sample plate, 10 ul of samples from
each well were picked by multi-channel pipette and dispensed in one
assay plate containing 40 ul molten diet in each well and mixed on
a shaker. This process of producing the assay plate was repeated as
many as 6 times or more to produce a desired number of assay
plates. After the diet was solidified and cooled to 4 C, neonate
insect larvae were placed in each well, sealed with perforated
Mylar film and incubated in a constant temperature incubator at
28.degree. C. After certain feeding period, the insect responses
were scored under a magnifying glass. The sigmoid dose-response
values (Responses) were converted to liner probit dose-response
values using SAS-JMP.RTM., Generalized Linear Model, Binomial
Response, Probit). The response for each protein in replicates was
summed and compared with the probit dose-response line of the
activity reference protein, creating a new number called the FAE
guide number (Fast Activity Evaluation). For example, if a mutant
protein showed a certain probit value at 40 ppm and the actual dose
with the same probit value for the reference protein was 100 ppm;
then the FAE value is 2.5 (100/40). This means the mutant protein
is 2.5 times more potent than the reference protein. This assay was
done with 2 different doses of mutant proteins at a time and
repeated 3 times generating 6 FAE guide number data points for each
mutant. The mean FAE guide number was called the FAE Index. For
each protein, a two sided t-test was done comparing the 6 FAE guide
numbers. The Bonferroni correction was used to evaluate p-values
(number of novel proteins/alpha) to determine if the FAE Index was
statistically significant.
[0226] The other screening method used in this patent application
is High Dose Assay (HDA). In this method, test proteins at high
concentrations (above EC50) were placed on the insect assay plates
as described above, along with a similar concentration of one or
more reference proteins with a known activity level. This HDA was
often used in a tiered screening to eliminate low or no activity
proteins quickly.
[0227] Yet another screening method used was High throughput
Functional Assay (HFA). This assay was similar to FAE but used only
one dose instead of 2 doses. Otherwise HFA, especially the way it
calculates the index was identical to FAE. Therefor the HFA index
has the same significance as the FAE index.
[0228] The predicted point with 50% response in the scoring scheme
is called ILC50 as it is a combination of growth or feeding
Inhibition and Lethal responses. In order to determine ILC50
values, each treatment (one dose) was repeated 6 or more, usually
24, times. The insecticidal activity of the Cry1B variants is shown
in Table 3.
[0229] Table 4 shows the insecticidal activity against corn earworm
for the amino acid substitutions having increased activity (FAE
score 1.2) compared to the reference polypeptide MP258 (SEQ ID NO:
47), IP1B-B21 (SEQ ID NO: 5), IP1B-B25 (SEQ ID NO: 13), or IP1B-B45
(SEQ ID NO: 41). Table 4 indicates the position number and amino
acid corresponding to positions 50-651 of MP258 (SEQ ID NO: 47);
the predicted secondary structure and assignment; solvent exposure
score; an alignment of the amino acid sequence of MP258 (SEQ ID NO:
47); IP1B-B21 (SEQ ID NO: 5), IP1B-B25 (SEQ ID NO: 13), IP1B-B45
(SEQ ID NO: 41), IP1B-B21 (SEQ ID NO: 5), Cry1Bd (SEQ ID NO: 1),
Cry1Bh (SEQ ID NO: 52), and Cry1Bi (SEQ ID NO: 54); the polypeptide
backbone the variant was made in; the amino acid substitution
variant (e.g. L50R); and the FAE insecticidal score against corn
earworm compared to the corresponding polypeptide backbone
(MP258--SEQ ID NO: 47, IP1B-B21--SEQ ID NO: 5, IP1B-B25--SEQ ID NO:
13, or IP1B-B45--SEQ ID NO: 41).
TABLE-US-00004 TABLE 3 Polypeptide Clone ID SEQ ID NO: ECB CEW FAW
Cry1Bd SEQ ID NO: 1 ILC50 = 1 ppm ILC50 = >1000 ppm ILC50 = ~400
ppm IP1B-B1 SEQ ID NO: 3 ILC50 = 1.3 ppm ILC50 = 21 ppm ILC50 =
34.3 ppm IP1B-B21 SEQ ID NO: 5 ILC50 = 22.4 ppm IP1B-B22 SEQ ID NO:
7 ILC50 = 27.1 ppm IP1B-B23 SEQ ID NO: 9 ILC50 = 29.2 ppm IP1B-B24
SEQ ID NO: 11 ILC50 = 12.6 ppm IP1B-B25 SEQ ID NO: 13 ILC50 = 11.91
ppm IP1B-B26 SEQ ID NO: 15 ILC50 = 8.36 ppm IP1B-B27 SEQ ID NO: 17
ILC50 = 7.99 ppm IP1B-B28 SEQ ID NO: 19 ILC50 = 7.74 ppm IP1B-B29
SEQ ID NO: 21 ILC50 = 8.45 ppm IP1B-B31 SEQ ID NO: 23 ILC50 = 2.8
ppm IP1B-B32 SEQ ID NO: 25 ILC50 = 2.9 ppm IP1B-B33 SEQ ID NO: 27
ILC50 = 3.0 ppm IP1B-B34 SEQ ID NO: 29 ILC50 = 2.9 ppm IP1B-B40 SEQ
ID NO: 31 ILC50 = 5.78 ppm IP1B-B41 SEQ ID NO: 33 ILC50 = 4.54 ppm
IP1B-B42 SEQ ID NO: 35 ILC50 = 6.2 ppm IP1B-B43 SEQ ID NO: 37 ILC50
= 6.7 ppm IP1B-B44 SEQ ID NO: 39 ILC50 = 6.9 ppm IP1B-B45 SEQ ID
NO: 41 ILC50 = 5.7 ppm IP1B-B46 SEQ ID NO: 43 ILC50 = 8 ppm
IP1B-B47 SEQ ID NO: 45 ILC50 = 6.1 ppm MP258 SEQ ID NO: 47 ILC50 =
4 ppm ILC50 = 24 ppm ILC50 = 62 ppm
[0230] Table 5 shows the insecticidal activity against corn earworm
for the amino acid substitutions having a FAE score 1.2 compared to
the polypeptide backbone MP258 (SEQ ID NO: 47), IP1B-B21 (SEQ ID
NO: 5), IP1B-B25 (SEQ ID NO: 13), or IP1B-B45 (SEQ ID NO: 41).
Table 5 indicates the position number and amino acid corresponding
to positions 50-651 of MP258 (SEQ ID NO: 47); the polypeptide
backbone the variant was made in; the amino acid substitution
variant (e.g. L50R); and the FAE insecticidal score against corn
earworm compared to the corresponding polypeptide backbone
(MP258--SEQ ID NO: 47, IP1B-B21--SEQ ID NO: 5, IP1B-B25--SEQ ID NO:
13, or IP1B-B45--SEQ ID NO: 41.
Example 5--Transient Expression in Maize Leaves and Insect
Bioassay
[0231] Polynucleotides encoding the variant Cry1B polypeptides were
cloned into transient expression vectors under control of the maize
ubiquitin promoter (Christensen and Quail, (1996) Transgenic
Research 5:213-218) and a duplicated version of the promoter from
the mirabilis mosaic virus (DMMV PRO; Dey and Maiti, (1999) Plant
Mol. Biol., 40:771-82). The agro-infiltration method of introducing
an Agrobacterium cell suspension to plant cells of intact tissues
so that reproducible infection and subsequent plant derived
transgene expression may be measured or studied is well known in
the art (Kapila, et. al., (1997) Plant Science 122:101-108).
Briefly, young plantlets of maize were agro-infiltrated with
normalized bacterial cell cultures of test and control strains.
Leaf discs were generated from each plantlet and infested with WCRW
(Diabrotica virgifera) along with appropriate controls. The degree
of consumption of green leaf tissues was scored after 2 days of
infestation.
Example 6--Transient Expression in Bush Bean Leaves and Insect
Bioassay
[0232] For soybean expression optimized coding sequences can be
designed. The agro-infiltration method of introducing an
Agrobacterium cell suspension to plant cells of intact tissues so
that reproducible infection and subsequent plant derived transgene
expression may be measured or studied is well known in the art
(Kapila, et. al., (1997) Plant Science 122:101-108). Briefly,
excised leaf disks of bush bean, are agro-infiltrated with
normalized bacterial cell cultures of test and control strains.
After 4 days leaf disks are infested with 2 neonates of Soybean
Looper (SBL) (Chrysodeixis includens), Corn Earworm, (CEW)
(Helicoverpa zea), Velvetbean Caterpillar (VBC) (Anticarsia
gemmatalis), or Fall Armyworm (Spodoptera frugiperda) alone.
Control leaf discs are generated with Agrobacterium containing only
a DsRed2 fluorescence marker (Clontech.TM., 1290 Terra Bella Ave.
Mountain View, Calif. 94043) expression vector. Leaf discs from
non-infiltrated plants are included as a second control. The
consumption of green leaf tissue is scored three days after
infestation and given scores of 0 to 9.
Example 7--Agrobacterium-Mediated Transformation of Maize and
Regeneration of Transgenic Plants
[0233] For Agrobacterium-mediated transformation of maize with a
polynucleotide sequence of the disclosure, the method of Zhao can
be used (U.S. Pat. No. 5,981,840 and PCT patent publication
WO98/32326; the contents of which are hereby incorporated by
reference). Briefly, immature embryos are isolated from maize and
the embryos contacted with a suspension of Agrobacterium under
conditions whereby the bacteria are capable of transferring the
toxin nucleotide sequence to at least one cell of at least one of
the immature embryos (step 1: the infection step). In this step the
immature embryos can be immersed in an Agrobacterium suspension for
the initiation of inoculation. The embryos are co-cultured for a
time with the Agrobacterium (step 2: the co-cultivation step). The
immature embryos can be cultured on solid medium following the
infection step. Following this co-cultivation period an optional
"resting" step is contemplated. In this resting step, the embryos
are incubated in the presence of at least one antibiotic known to
inhibit the growth of Agrobacterium without the addition of a
selective agent for plant transformants (step 3: resting step). The
immature embryos can be cultured on solid medium with antibiotic,
but without a selecting agent, for elimination of Agrobacterium and
for a resting phase for the infected cells. Next, inoculated
embryos are cultured on medium containing a selective agent and
growing transformed callus is recovered (step 4: the selection
step). The immature embryos are cultured on solid medium with a
selective agent resulting in the selective growth of transformed
cells. The callus is then regenerated into plants (step 5: the
regeneration step), and calli grown on selective medium can be
cultured on solid medium to regenerate the plants.
Example 8--Transformation of Soybean Embryos
[0234] Soybean embryos are bombarded with a plasmid containing the
toxin nucleotide sequence operably linked to a suitable promoter as
follows. To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface-sterilized, immature seeds of an appropriate
soybean cultivar are cultured in the light or dark at 26.degree. C.
on an appropriate agar medium for six to ten weeks. Somatic embryos
producing secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos that multiplied as early, globular-staged embryos,
the suspensions are maintained as described below.
[0235] Soybean embryogenic suspension cultures can be maintained in
35 mL liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 mL of liquid medium.
[0236] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein, et
al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0237] A selectable marker gene that can be used to facilitate
soybean transformation includes, but is not limited to: the 35S
promoter from Cauliflower Mosaic Virus (Odell, et al., (1985)
Nature 313:810-812), the hygromycin phosphotransferase gene from
plasmid pJR225 (from E. coli; Gritz, et al., (1983) Gene
25:179-188), and the 3' region of the nopaline synthase gene from
the T DNA of the Ti plasmid of Agrobacterium tumefaciens. The
expression cassette comprising a toxin nucleotide sequence (e.g.,
SEQ ID NO: 1, SEQ ID NO: 3 or a maize optimized sequence) operably
linked to a suitable promoter can be isolated as a restriction
fragment. This fragment can then be inserted into a unique
restriction site of the vector carrying the marker gene.
[0238] To 50 .mu.L of a 60 mg/mL 1 .mu.m gold particle suspension
is added (in order): 5 .mu.L DNA (1 .mu.g/.mu.L), 20 .mu.L
spermidine (0.1M), and 50 .mu.L CaCl2 (2.5M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.L 70% ethanol and
resuspended in 40 .mu.L of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0239] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi,
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0240] Five to seven days post bombardment the liquid media may be
exchanged with fresh media, and eleven to twelve days
post-bombardment with fresh media containing 50 mg/mL hygromycin.
This selective media can be refreshed weekly. Seven to eight weeks
post-bombardment, green, transformed tissue may be observed growing
from untransformed, necrotic embryogenic clusters. Isolated green
tissue is removed and inoculated into individual flasks to generate
new, clonally propagated, transformed embryogenic suspension
cultures. Each new line may be treated as an independent
transformation event. These suspensions can then be subcultured and
maintained as clusters of immature embryos or regenerated into
whole plants by maturation and germination of individual somatic
embryos.
[0241] All publications, patents and patent applications mentioned
in the specification are indicative of the level of those skilled
in the art to which this disclosure pertains. All publications,
patents and patent applications are herein incorporated by
reference to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated by reference.
[0242] Although the foregoing disclosure has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the embodiments.
Sequence CWU 1
1
611657PRTBacillus thuringiensis 1Met Thr Ser Asn Arg Lys Asn Glu
Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn
His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala Arg Ile Glu
Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40 45 Pro Leu
Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60
Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser 65
70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg
Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile
Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala
Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln
Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser
Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala 145 150 155 160 Leu Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile Arg Asn 165 170 175 Glu
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met
195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg
Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser
Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile
Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe
Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310
315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro
Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser
Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Asn Phe
Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr Gln Gly Leu
Thr Asn Asn Thr Ser Ile Asn 370 375 380 Pro Val Thr Leu Gln Phe Thr
Ser Arg Asp Val Tyr Arg Thr Glu Ser 385 390 395 400 Asn Ala Gly Thr
Asn Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro 405 410 415 Trp Ala
Arg Phe Asn Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly 420 425 430
Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe 435
440 445 Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr 450 455 460 Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile
Ile Gly Asn 465 470 475 480 Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg 485 490 495 Thr Asn Thr Ile Gly Pro Asn Arg
Ile Thr Gln Ile Pro Ala Val Lys 500 505 510 Gly Arg Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr 515 520 525 Gly Gly Asp Val
Val Arg Leu Asn Arg Asn Asn Gly Asn Ile Gln Asn 530 535 540 Arg Gly
Tyr Ile Glu Val Pro Ile Gln Phe Thr Ser Thr Ser Thr Arg 545 550 555
560 Tyr Arg Val Arg Val Arg Tyr Ala Ser Val Thr Ser Ile Glu Leu Asn
565 570 575 Val Asn Leu Gly Asn Ser Ser Ile Phe Thr Asn Thr Leu Pro
Ala Thr 580 585 590 Ala Ala Ser Leu Asp Asn Leu Gln Ser Gly Asp Phe
Gly Tyr Val Glu 595 600 605 Ile Asn Asn Ala Phe Thr Ser Ala Thr Gly
Asn Ile Val Gly Ala Arg 610 615 620 Asn Phe Ser Ala Asn Ala Glu Val
Ile Ile Asp Arg Phe Glu Phe Ile 625 630 635 640 Pro Val Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln 645 650 655 Lys
21971DNABacillus thuringiensis 2atgccttcaa ataggaaaaa tgagaatgaa
attataaatg ccttatcgat tccagctgta 60tcgaatcatt ccgcacaaat ggatctatcg
ctagatgctc gtattgagga ttctttgtgt 120atagccgagg ggaataatat
caatccactt gttagcgcat caacagtcca aacgggtata 180aacatagctg
gtagaatatt gggcgtatta ggtgtgccgt ttgctggaca actagctagt
240ttttatagtt ttcttgttgg ggaattatgg cctagtggca gagatccatg
ggaaattttc 300ctggaacatg tagaacaact tataagacaa caagtaacag
aaaatactag gaatacggct 360attgctcgat tagaaggtct aggaagaggc
tatagatctt accagcaggc tcttgaaact 420tggttagata accgaaatga
tgcaagatca agaagcatta ttcttgagcg ctatgttgct 480ttagaacttg
acattactac tgctataccg cttttcagaa tacgaaatga agaagttcca
540ttattaatgg tatatgctca agctgcaaat ttacacctat tattattgag
agacgcatcc 600ctttttggta gtgaatgggg gatggcatct tccgatgtta
accaatatta ccaagaacaa 660atcagatata cagaggaata ttctaaccat
tgcgtacaat ggtataatac agggctaaat 720aacttaagag ggacaaatgc
tgaaagttgg ttgcggtata atcaattccg tagagaccta 780acgttagggg
tattagattt agtagcccta ttcccaagct atgatactcg cacttatcca
840atcaatacga gtgctcagtt aacaagagaa atttatacag atccaattgg
gagaacaaat 900gcaccttcag gatttgcaag tacgaattgg tttaataata
atgcaccatc gttttctgcc 960atagaggctg ccattttcag gcctccgcat
ctacttgatt ttccagaaca acttacaatt 1020tacagtgcat caagccgttg
gagtagcact caacatatga attattgggt gggacatagg 1080cttaacttcc
gcccaatagg agggacatta aatacctcaa cacaaggact tactaataat
1140acttcaatta atcctgtaac attacagttt acgtctcgtg acgtttatag
aacagaatca 1200aatgcaggga caaatatact atttactact cctgtgaatg
gagtaccttg ggctagattt 1260aattttataa accctcagaa tatttatgaa
agaggcgcca ctacctacag tcaaccgtat 1320cagggagttg ggattcaatt
atttgattca gaaactgaat taccaccaga aacaacagaa 1380cgaccaaatt
atgaatcata tagtcataga ttatctcata taggactaat cataggaaac
1440actttgagag caccagtcta ttcttggacg catcgtagtg cagatcgtac
gaatacgatt 1500ggaccaaata gaattactca aattcctgca gtgaagggaa
gatttctttt taatggttct 1560gtaatttcag gaccaggatt tactggtgga
gacgtagtta gattgaatag gaataatggt 1620aatattcaaa atagagggta
tattgaagtt ccaattcaat tcacgtcgac atctaccaga 1680tatcgagttc
gagtacgtta tgcttctgta acctcgattg agctcaatgt taatttgggc
1740aattcatcaa tttttacgaa cacattacca gcaacagctg catcattaga
taatctacaa 1800tcaggggatt ttggttatgt tgaaatcaac aatgctttta
catccgcaac aggtaatata 1860gtaggtgcta gaaattttag tgcaaatgca
gaagtaataa tagacagatt tgaatttatc 1920ccagttactg caaccttcga
ggcagaatat gatttagaaa gagcacaaaa g 19713651PRTArtificial
SequenceCry1B variant 3Met Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile
Ile Asn Ala Val Ser 1 5 10 15 Asn His Ser Ala Gln Met Asp Leu Ser
Leu Asp Ala Arg Ile Glu Asp 20 25 30 Ser Leu Cys Val Ala Glu Val
Asn Asn Ile Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val Gln Thr
Gly Ile Ser Ile Ala Gly Arg Ile Leu Gly Val 50 55 60 Leu Gly Val
Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val
Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro Trp Glu Ile Phe Met 85 90
95 Glu His Val Glu Gln Ile Val Arg Gln Gln Ile Thr Asp Ser Val Arg
100 105 110 Asp Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly Arg Gly Tyr
Arg Ser 115 120 125 Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg
Asn Asp Ala Arg 130 135 140 Ser Arg Ser Ile Ile Arg Glu Arg Tyr Ile
Ala Leu Glu Leu Asp Ile 145 150 155 160 Thr Thr Ala Ile Pro Leu Phe
Ser Ile Arg Asn Gln Glu Val Pro Leu 165 170 175 Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190 Asp Ala Ser
Leu Phe Gly Ser Glu Trp Gly Met Ser Ser Ser Asp Val 195 200 205 Asn
Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr Glu Glu Tyr Ser Asn 210 215
220 His Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr
225 230 235 240 Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg
Asp Leu Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro
Ser Tyr Asp Thr Arg 260 265 270 Val Tyr Pro Met Asn Thr Ser Ala Gln
Leu Thr Arg Glu Ile Tyr Thr 275 280 285 Asp Pro Ile Gly Arg Thr Asn
Ala Pro Ser Gly Phe Ala Ser Thr Asn 290 295 300 Trp Phe Asn Asn Asn
Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ile 305 310 315 320 Phe Arg
Pro Pro His Leu Leu Asp Phe Pro Glu Gln Leu Thr Ile Tyr 325 330 335
Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His Met Asn Tyr Trp Val 340
345 350 Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr
Ser 355 360 365 Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr
Leu Gln Phe 370 375 380 Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe
Ala Gly Thr Asn Ile 385 390 395 400 Leu Phe Thr Thr Pro Val Asn Gly
Val Pro Trp Ala Arg Phe Asn Phe 405 410 415 Ile Asn Pro Gln Asn Ile
Tyr Glu Arg Gly Ala Thr Thr Tyr Ser Gln 420 425 430 Pro Tyr Gln Gly
Val Gly Ile Gln Leu Phe Asp Ser Glu Thr Glu Leu 435 440 445 Pro Pro
Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg 450 455 460
Leu Ser His Ile Gly Leu Ile Ile Gly Asn Thr Leu Arg Ala Pro Val 465
470 475 480 Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile
Gly Pro 485 490 495 Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Gly Arg
Phe Leu Phe Asn 500 505 510 Gly Ser Val Ile Ser Gly Pro Gly Phe Thr
Gly Gly Asp Val Val Arg 515 520 525 Leu Asn Arg Asn Asn Gly Asn Ile
Gln Asn Arg Gly Tyr Ile Glu Val 530 535 540 Pro Ile Gln Phe Thr Ser
Thr Ser Thr Arg Tyr Arg Val Arg Val Arg 545 550 555 560 Tyr Ala Ser
Val Thr Ser Ile Glu Leu Asn Val Asn Leu Gly Asn Ser 565 570 575 Ser
Ile Phe Thr Asn Thr Leu Pro Ala Thr Ala Ala Ser Leu Asp Asn 580 585
590 Leu Gln Ser Gly Asp Phe Gly Tyr Val Glu Ile Asn Asn Ala Phe Thr
595 600 605 Ser Ala Thr Gly Asn Ile Val Gly Ala Arg Asn Phe Ser Ala
Asn Ala 610 615 620 Glu Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val
Thr Ala Thr Phe 625 630 635 640 Glu Ala Glu Tyr Asp Leu Glu Arg Ala
Gln Lys 645 650 41953DNAArtificial Sequencecoding sequence of Cry1B
variant 4atgccttcaa ataggaaaaa tgagaatgaa attataaatg ctgtatcgaa
tcattccgca 60caaatggatc tatcgctaga tgctcgtatt gaagatagct tgtgtgtagc
cgaggtgaac 120aatattgatc catttgttag cgcatcaaca gtccaaacag
gtattagtat agctggtaga 180atattgggcg tattaggtgt gccgtttgct
ggacaactag ctagttttta tagttttctt 240gttggggaat tatggcctag
cggcagagat ccatgggaaa tttttatgga acatgtcgag 300caaattgtaa
gacaacaaat aacggacagt gttagggata ccgctattgc tcgtttagaa
360ggtctaggaa gagggtatag atcttaccag caggctcttg aaacttggtt
agataaccga 420aatgatgcaa gatcaagaag cattattcgt gagagatata
ttgctttaga acttgacatt 480actactgcta taccgctttt cagcatacga
aatcaagagg ttccattatt aatggtatat 540gctcaagctg caaatttaca
cctattatta ttgagagacg catccctttt tggtagtgaa 600tgggggatgt
catcttccga tgttaaccaa tattaccaag aacaaatcag atatacagag
660gaatattcta accattgcgt acaatggtat aatacagggc taaataactt
aagagggaca 720aatgctgaaa gttggttgcg gtataatcaa ttccgtagag
atctaacgtt aggagtatta 780gatctagtgg cactattccc aagctatgac
acgcgtgttt atccaatgaa taccagtgct 840caattaacaa gagaaattta
tacagatcca attgggagaa caaatgcacc ttcaggattt 900gcaagtacga
attggtttaa taataatgca ccatcgtttt ctgccataga ggctgccatt
960ttcaggcctc cgcatctact tgattttcca gaacaactta caatttacag
tgcatcaagc 1020cgttggagta gcactcaaca tatgaattat tgggtgggac
ataggcttaa cttccgccca 1080ataggaggga cattaaatac ctcaacgcat
ggggctacca atacttctat taatcctgta 1140acattacagt tcacatctcg
agacgtttat aggactgaat catttgcagg gacaaatata 1200ctatttacta
ctcctgtgaa tggagtacct tgggctagat ttaattttat aaaccctcag
1260aatatttatg aaagaggcgc cactacctac agtcaaccgt atcagggagt
tgggattcaa 1320ttatttgatt cagaaactga attaccacca gaaacaacag
aacgaccaaa ttatgaatca 1380tatagtcata gattatctca tataggacta
atcataggaa acactttgag agcaccagtc 1440tattcttgga cgcaccgtag
tgcagatcgt acgaatacga ttggaccaaa tagaattact 1500caaattcctg
cagtgaaggg aagatttctt tttaatggtt ctgtaatttc aggaccagga
1560tttactggtg gagacgtagt tagattgaat aggaataatg gtaatattca
aaatagaggg 1620tatattgaag ttccaattca attcacgtcg acatctacca
gatatcgagt tcgagtacgt 1680tatgcttctg taacctcgat tgagctcaat
gttaatttgg gcaattcatc aatttttacg 1740aacacattac cagcaacagc
tgcatcatta gataatctac aatcagggga ttttggttat 1800gttgaaatca
acaatgcttt tacatccgca acaggtaata tagtaggtgc tagaaatttt
1860agtgcaaatg cagaagtaat aatagacaga tttgaattta tcccagttac
tgcaaccttc 1920gaggcagaat atgatttaga aagagcacaa aag
19535655PRTArtificial SequenceCry1B variant 5Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Gln Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355
360 365 Thr Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn
Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr
Glu Ser Tyr 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro
Val Asn Gly Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro
Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr
Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu
Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser
His Arg Leu Ser Asn Ile Arg Leu Ile Ile Gly Asn Thr Leu 465 470 475
480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn
485 490 495 Thr Ile Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala Val Lys
Gly Asn 500 505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly
Phe Thr Gly Gly 515 520 525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn
Asn Ile Gln Asn Arg Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe
Ile Ser Thr Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr
Ala Ser Val Thr Pro Ile Gln Leu Ser Val Asn 565 570 575 Trp Gly Asn
Ser Asn Ile Phe Ser Ser Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser
Leu Asp Asn Leu Gln Ser Arg Asn Phe Gly Tyr Phe Glu Ser Thr 595 600
605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe
610 615 620 Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile
Pro Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu
Arg Ala Gln Glu 645 650 655 61965DNAArtificial Sequencecoding
sequence of Cry1B variant 6atgccgagca atcgtaagaa tgaaaatgaa
atcattaacg cactgtccat ccctgcagtg 60agcaatcaca gcgcgcagat ggatttgagc
ctggatgcgc gtatcgagga cagcctgtgt 120attgccgagg gcaacaacat
caatccgttg gtcagcgcga gcaccgtgca aaccggcatt 180aacattgccg
gtcgtatcct gggtgtcctg ggcgttccgt ttgcgggtca gctggcgagc
240ttttacagct ttatcgttgg tgagttgtgg ccgtcgggtc gtgacccttg
ggagattttc 300atggagcacg tcgagcaact ggtgcgccaa cagattacgg
agaatgcgcg caacaccgct 360ctggcgcgtc tgcaaggtct gggtgcaagc
ttccgcgctt accagcagtc cctggaagat 420tggttggaaa accgtgataa
tgcgcgcact cgctccgtcc tgtacacgca gtacatcgcg 480ctggagctgg
acttcttgaa cgcgatgccg ctgtttgcaa tcaacaacca gcaagtgccg
540ctgctgatgg tctacgccca agccgcgaat ctgcacttgc tgctgctgcg
cgacgcatct 600ctgttcggta gcgaatttgg cctgaccagc caggagatcc
agcgctacta tgagcgtcag 660gccgagaaaa cgcgtgaata ctccgactac
tgcgctcgtt ggtacaacac gggtctgaac 720aatctgcgtg gcaccaacgc
ggagtcctgg ctgcgttaca accagtttcg tcgcgatctg 780accctgggtg
ttttggattt ggttgcgctg tttccgagct atgacacccg catctatccg
840atcaacacca gcgcgcaact gactcgtgaa atctatacgg acccgattgg
ccgcactaat 900gcaccgtccg gtttcgcaag caccaactgg ttcaataaca
atgcaccgag cttcagcgcg 960atcgaggccg cgatctttcg tccgccgcac
ctgttggact tcccggagca gctgaccatc 1020tactctgcat ctagccgttg
gagcagcacg cagcacatga attactgggt tggccatcgt 1080ctgaacttcc
gcccgattgg tggtacgctg aacactagca cgcacggtgc cactaacacg
1140agcatcaacc cggtgacgct gcaattcacc agccgtgatg tttaccgtac
cgagtcctac 1200gccggcatca acattctgct gaccaccccg gttaacggcg
tcccttgggc tcgtttcaat 1260tggcgtaacc cactgaatag cctgcgtggt
tctttgctgt acaccattgg ttataccggc 1320gtcggtacgc aactgtttga
ctcggaaact gagctgccac cggaaactac cgagcgtccg 1380aactacgaat
cttatagcca ccgtctgtcc aatatccgtc tgatcatcgg caacaccctg
1440cgtgcgccgg tgtacagctg gacccatcgt agcgccgatc gcacgaacac
gattgccacc 1500aacattatca cccagatccc ggcagtgaaa ggcaactttc
tgtttaacgg cagcgtgatc 1560agcggtccag gttttaccgg cggtgacctg
gtgcgcctga acaacagcgg caacaatatc 1620caaaaccgtg gttatatcga
agtcccgatt caattcatca gcacgagcac ccgttaccgc 1680gtccgtgttc
gctacgcatc cgttacgccg atccaactga gcgttaactg gggcaattcc
1740aacattttca gcagcattgt ccctgctacg gcgacctctc tggacaattt
gcagagccgt 1800aacttcggct atttcgaaag caccaacgct ttcaccagcg
ctacgggcaa tgtggttggt 1860gttcgcaatt tcagcgagaa tgcgggcgtc
atcattgacc gttttgagtt tatcccggtg 1920accgcgacct tcgaagcgga
gtacgatctg gagcgtgcgc aggaa 19657655PRTArtificial SequenceCry1B
variant 7Met Pro Ser Asn Arg Lys Asn Glu Asn Gly Ile Ile Asn Ala
Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asp
Leu Ser Pro Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Val Ala
Glu Val Asn Asn Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val
Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu
Gly Val Pro Phe Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser Phe
Ile Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu
Ile Phe Leu Glu His Val Glu Gln Leu Val Arg Gln Gln Ile 100 105 110
Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115
120 125 Ala Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu
Asn 130 135 140 Arg Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln
Tyr Ile Ala 145 150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro
Leu Phe Ala Ile Asn Asn 165 170 175 Gln Arg Val Pro Leu Leu Met Val
Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp
Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu
Ile Gln Arg Tyr Tyr Glu Arg Gln Ala Glu Lys Thr 210 215 220 Arg Glu
Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235
240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe
245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu
Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Val Tyr Pro Ile Asn Thr Ser
Ala Gln Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg
Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn
Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val
Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr
Ile Phe Ser Val Leu Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met
Asn Tyr Trp Val Gly His Arg Leu Glu Ser Arg Thr Ile Arg Gly 355 360
365 Ser Leu Ser Thr Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro
370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu
Ser Tyr 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val
Asn Gly Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu
Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr
Gly Val Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro
Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His
Arg Leu Ser His Ile Gly Leu Ile Ile Gly Asn Thr Leu 465 470 475 480
Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485
490 495 Thr Thr Gly Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Gly
Asn 500 505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe
Thr Gly Gly 515 520 525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn Asn
Ile Gln Asn Arg Gly 530 535 540 Tyr Leu Glu Val Pro Ile Gln Phe Ile
Ser Thr Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala
Ser Val Thr Pro Ile Gln Leu Ser Val Asn 565 570 575 Trp Gly Asn Ser
Asn Ile Phe Ser Ser Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser Leu
Asp Asn Leu Gln Ser Arg Asp Phe Gly Tyr Phe Glu Ser Thr 595 600 605
Asn Ala Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe 610
615 620 Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro
Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg
Ala Gln Glu 645 650 655 81965DNAArtificial Sequencecoding sequence
of Cry1B variant 8atgccgagca atcgtaagaa tgaaaatgga atcattaacg
cgctgtccat ccctgcagtg 60agcaatcaca gcgcgcagat ggatttgagc ccggatgcgc
gtatcgagga cagcctgtgt 120gtcgccgagg taaacaatat tgatccgttc
gtcagcgcga gcaccgtgca aaccggcatt 180aacattgccg gtcgtatcct
gggtgtcctg ggcgttccgt ttgcgggtca gctggcgagc 240ttttacagct
ttatcgttgg tgagttgtgg ccgtcgggtc gtgacccttg ggagattttc
300ttggagcacg tcgagcaact ggtgcgccaa cagattacgg agaatgcgcg
caacaccgct 360ctggcgcgtc tgcaaggtct gggtgcaagc ttccgcgctt
accagcagtc cctggaagat 420tggttggaaa accgtgatga tgcgcgcact
cgctccgtcc tgtacacgca gtacatcgcg 480ctggagctgg acttcttgaa
cgcgatgccg ctgtttgcaa tcaacaacca gcgagtgccg 540ctgctgatgg
tctacgccca agccgcgaat ctgcacttgc tgctgctgcg cgacgcatct
600ctgttcggta gcgaatttgg cctgaccagc caggagatcc agcgctacta
tgagcgtcag 660gccgagaaaa cgcgtgaata ctccgactac tgcgctcgtt
ggtacaacac gggtctgaac 720aatctgcgtg gcaccaacgc ggagtcctgg
ctgcgttata accagtttcg tcgcgatctg 780accctgggtg tattggattt
ggttgcgctg tttccgagct atgacacccg cgtgtatccg 840atcaacacca
gcgcgcaact gactcgtgaa atctatacgg acccgattgg ccgcactaat
900gcaccgtccg gtttcgcaag caccaactgg ttcaataaca atgcaccgag
cttcagcgcg 960atcgaggcgg ctgtcatccg tccgccgcac ctgttggact
tcccggagca gctgaccatc 1020ttttctgtgt tgtctcgttg gagcagcacg
cagcacatga attactgggt tggccatcgt 1080ctggaaagcc gcaccattcg
cggtagcctg agcactagca cgcacggtaa tactaacacg 1140agcatcaacc
cggtgacgct gcaattcacc agccgtgatg tttaccgtac cgagtcctac
1200gccggcatca acattctgct gaccaccccg gttaacggcg tcccttgggc
tcgtttcaat 1260tggcgtaacc cactgaatag cctgcgtggt tctttgctgt
acaccattgg ttataccggc 1320gtcggtacgc aactgtttga ctcggaaact
gagctgccac cggaaactac cgagcgtccg 1380aactacgaat cttatagcca
ccgtctgtcc catattggtc tgatcatcgg caacaccctg 1440cgtgcaccgg
tgtacagctg gacccatcgt agcgccgatc gcacgaacac gactggtccg
1500aaccgtatca cccagatccc ggcagtgaaa ggcaactttc tgtttaacgg
cagcgtgatc 1560agcggtccag gttttaccgg cggtgacctg gtgcgcctga
acaacagcgg caacaatatc 1620caaaaccgtg gttatctgga agtcccgatt
caattcatca gcacgagcac ccgttaccgc 1680gtccgtgttc gctacgcatc
cgttacgccg atccaactga gcgttaactg gggcaattcc 1740aacattttca
gcagcattgt ccctgctacg gcgacctctc tggacaattt gcagagccgt
1800gacttcggct atttcgaaag caccaacgct ttcaccagcg ctacgggcaa
tgtggttggt 1860gttcgcaatt tcagcgagaa tgcgggcgtc atcattgacc
gttttgagtt tatcccggtg 1920accgcgacct tcgaagcgga gtacgatctg
gagcgtgcgc aggaa 19659650PRTArtificial SequenceCry1B variant 9Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser 1 5 10
15 Asn His Ser Ala Gln Met Asp Leu Ser Pro Asp Ala Arg Ile Glu Asp
20 25 30 Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp Pro Phe Val
Ser Ala 35 40 45 Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly Arg
Ile Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gln Leu Ala
Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Ser Gly
Arg Asp Pro Trp Glu Ile Phe Met 85 90 95 Glu His Val Glu Gln Ile
Val Arg Gln Gln Ile Thr Glu Asn Ala Arg 100 105 110 Asn Thr Ala Leu
Ala Arg Leu Gln Gly Leu Gly Ala Ser Phe Arg Ala 115 120 125 Tyr Gln
Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp Ala Arg 130 135 140
Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe 145
150 155 160 Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn Gln Gln Val
Pro Leu 165 170 175 Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu
Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly
Leu Thr Ser Gln Glu Ile 195 200 205 Gln Arg Tyr Tyr Glu Arg Gln Ala
Glu Lys Thr Arg Glu Tyr Ser Asp 210 215 220 Tyr Cys Ala Arg Trp Tyr
Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr 225 230 235 240 Asn Ala Glu
Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr 245 250 255 Leu
Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265
270 Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Ile Tyr Thr
275 280 285 Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly Phe Ala Ser
Thr Asn 290 295 300 Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile
Glu Ala Ala Val 305 310 315 320 Ile Arg Pro Pro His Leu Leu Asp Phe
Pro Glu Gln Leu Thr Ile Tyr 325 330 335 Ser Ala Ser Ser Arg Trp Ser
Ser Thr Gln His Met Asn Tyr Trp Val 340 345 350 Gly His Arg Leu Asn
Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365 Thr His Gly
Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Gln Phe 370 375 380 Thr
Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Ile Asn Ile 385 390
395 400 Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe Asn
Trp 405 410 415 Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu Leu Tyr
Thr Ile Gly 420 425 430 Tyr Thr Gly Val Gly Ile Gln Leu Phe Asp Ser
Glu Thr Glu Leu Pro 435 440 445 Pro Glu Thr Thr Glu Arg Pro Asn Tyr
Glu Ser Tyr Ser His Arg Leu 450 455 460 Ser Asn Ile Arg Leu Ile Ser
Gly Asn Thr Leu Arg Ala Pro Val Tyr 465 470 475 480 Ser Trp Thr His
Arg Ser Ala Asp Arg Thr Asn Thr Ile Ala Thr Asn 485 490 495 Ile Ile
Thr Gln Ile Pro Ala Val Lys Gly Asn Phe Leu Phe Asn Gly 500 505 510
Ser Val Thr Ser Gly Pro Gly Phe Thr Gly Gly Asp Leu Val Arg Leu 515
520 525 Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly Tyr Leu Glu Val
Pro 530 535 540 Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg Val Arg
Val Arg Tyr 545 550 555 560 Ala Ser Val Thr Pro Ile Gln Leu Ser Val
Asn Trp Gly Asn Ser Asn 565 570 575 Ile Phe Ser Ser Ile Val Pro Ala
Thr Ala Thr Ser Leu Asp Asn Leu 580 585 590 Gln Ser Arg Asp Phe Gly
Tyr Phe Glu Ser Thr Asn Ala Phe Thr Ser 595 600 605 Ala Thr Gly Asn
Val Val Gly Val Arg Asn Phe Ser Glu Asn Ala Gly 610 615 620 Val Ile
Ile Asp Arg Phe Glu Phe Ile Pro Val Thr Ala Thr Phe Glu 625 630 635
640 Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650
101950DNAArtificial Sequencecoding sequence of Cry1B variant
10atgccgagca atcgtaagaa tgaaaatgaa atcattaacg cagtgagcaa tcacagcgcg
60cagatggatt tgagcccgga tgcgcgtatc gaggacagcc tgtgtgtcgc cgaggtaaac
120aatattgatc cgttcgtcag cgcgagcacc gtgcaaaccg gcattaacat
tgccggtcgt 180atcctgggtg tcctgggcgt tccgtttgcg ggtcagctgg
cgagctttta cagctttttg 240gttggtgagt tgtggccgtc gggtcgtgac
ccttgggaga ttttcatgga gcacgtcgag 300caaattgtgc gccaacagat
tacggagaat gcgcgcaaca ccgctctggc gcgtctgcaa 360ggtctgggtg
caagcttccg cgcttaccag cagtccctgg aagattggtt
ggaaaaccgt 420gatgatgcgc gcactcgctc cgtcctgtac acgcagtaca
tcgcgctgga gctggacttc 480ttgaacgcga tgccgctgtt tgcaatcaac
aaccagcaag tgccgctgct gatggtctac 540gcccaagccg cgaatctgca
cttgctgctg ctgcgcgacg catctctgtt cggtagcgaa 600tttggcctga
ccagccagga gatccagcgc tactatgagc gtcaggccga gaaaacgcgt
660gaatactccg actactgcgc tcgttggtac aacacgggtc tgaacaatct
gcgtggcacc 720aacgcggagt cctggctgcg ttacaaccag tttcgtcgcg
atctgaccct gggtgttttg 780gatttggttg cgctgtttcc gagctatgac
acccgcatct atccgatcaa caccagcgcg 840caactgactc gtgaaatcta
tacggacccg attggccgca ctaatgcacc gtccggtttc 900gcaagcacca
actggttcaa taacaatgca ccgagcttca gcgcgatcga ggcggctgtc
960atccgtccgc cgcacctgtt ggacttcccg gagcagctga ccatctactc
tgcatctagc 1020cgttggagca gcacgcagca catgaattac tgggttggcc
atcgtctgaa cttccgcccg 1080attggtggta cgctgaacac tagcacgcac
ggtgccacta acacgagcat caacccggtg 1140acgctgcaat tcaccagccg
tgatgtttac cgtaccgagt cctacgccgg gatcaacatt 1200ctgctgacca
ccccggttaa cggcgtccct tgggctcgtt tcaattggcg taacccactg
1260aatagcctgc gtggttcttt gctgtacacc attggttata ccggcgtcgg
tattcaactg 1320tttgactcgg aaactgagct gccaccggaa actaccgagc
gtccgaacta cgaatcttat 1380agccaccgtc tgtccaatat ccgtctgatc
agcggcaaca ccctgcgtgc gccggtgtac 1440agctggaccc accgtagcgc
cgatcgcacg aacacgattg ccaccaacat tatcacccag 1500atcccggcag
tgaaaggcaa ctttctgttt aacggcagcg tgaccagcgg tccaggtttt
1560accggcggtg acctggtgcg cctgaacaac agcggcaaca atatccaaaa
ccgtggttat 1620ctggaagtcc cgattcaatt catcagcacg agcacccgtt
accgcgtccg tgttcgctac 1680gcatccgtta cgccgatcca actgagcgtt
aactggggca attccaacat tttcagcagc 1740attgtccctg ctacggcgac
ctctctggac aatttgcaga gccgtgactt cggctatttc 1800gaaagcacca
acgctttcac cagcgctacg ggcaatgtgg ttggtgttcg caatttcagc
1860gagaatgcgg gcgtcatcat tgaccgtttt gagtttatcc cggtgaccgc
gaccttcgaa 1920gcggagtacg atctggagcg tgcgcaggaa
195011655PRTArtificial SequenceCry1B variant 11Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Gln Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Gln Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Arg Ile Val Pro
Ala Thr Ala Tyr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Thr 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
121965DNAArtificial Sequencecoding sequence of Cry1B variant
12atgccgagca atcgtaagaa tgaaaatgaa atcattaacg cactgtccat ccctgcagtg
60agcaatcaca gcgcgcagat ggatttgagc ctggatgcgc gtatcgagga cagcctgtgt
120attgccgagg gcaacaacat caatccgttg gtcagcgcga gcaccgtgca
aaccggcatt 180aacattgccg gtcgtatcct gggtgtcctg ggcgttccgt
ttgcgggtca gctggcgagc 240ttttacagct ttatcgttgg tgagttgtgg
ccgtcgggtc gtgacccttg ggagattttc 300atggagcacg tcgagcaact
ggtgcgccaa cagattacgg agaatgcgcg caacaccgct 360ctggcgcgtc
tgcaaggtct gggtgcaagc ttccgcgctt accagcagtc cctggaagat
420tggttggaaa accgtgataa tgcgcgcact cgctccgtcc tgtacacgca
gtacatcgcg 480ctggagctgg acttcttgaa cgcgatgccg ctgtttgcaa
tcaacaacca gcaagtgccg 540ctgctgatgg tctacgccca agccgcgaat
ctgcacttgc tgctgctgcg cgacgcatct 600ctgttcggta gcgaatttgg
cctgaccagc caggagatcc agcgctacta tgagcgtcag 660gccgagaaaa
cgcgtgaata ctccgactac tgcgctcgtt ggtacaacac gggtctgaac
720aatctgcgtg gcaccaacgc ggagtcctgg ctgcgttaca accagtttcg
tcgcgatctg 780accctgggtg ttttggattt ggttgcgctg tttccgagct
atgacacccg catctatccg 840atcaacacca gcgcgcaact gactcgtgaa
atctatacgg acccgattgg ccgcactaat 900gcaccgtccg gtttcgcaag
caccaactgg ttcaataaca atgcaccgag cttcagcgcg 960atcgaggccg
cgatctttcg tccgccgcac ctgttggact tcccggagca gctgaccatc
1020tactctgcat ctagccgttg gagcagcacg cagcacatga attactgggt
tggccatcgt 1080ctgaacttcc gcccgattgg tggtacgctg aacactagca
cgcacggtgc cactaacacg 1140agcatcaacc cggtgacgct gcaattcacc
agccgtgatg tttaccgtac cgagtcctac 1200gccggcatca acattctgct
gaccaccccg gttaacggcg tcccttgggc tcgtttcaat 1260tggcgtaacc
cactgaatag cctgcgtggt tctttgctgt acaccattgg ttataccggc
1320gtcggtacgc aactgtttga ctcggaaact gagctgccac cggaaactac
cgagcgtccg 1380aactacgaat cttatagcca ccgtctgtcc aatatccgtc
tgatcatcgg caacaccctg 1440cgtgcgccgg tgtacagctg gacccatcgt
agcgccgatc gcacgaacac gattgccacc 1500aacattatca cccagatccc
ggcagtgaaa ggcaactttc tgtttaacgg cagcgtgatc 1560agcggtccag
gttttaccgg cggtgacctg gtgcgcctga acaacagcgg caacaatatc
1620caaaaccgtg gttatatcga agtcccgatt caattcatca gcacgagcac
ccgttaccgc 1680gtccgtgttc gctacgcatc cgttacgccg atccaactga
gcgttaactg gggcaattcc 1740aacattttca gccgcattgt ccctgctacg
gcgtactctc tggacaattt gcagagccgt 1800aacttcggct atttcgaaag
caccaacgct ttcaccagcg ctacgggcaa tgtggttggt 1860gttcgcaatt
tcagcgagaa tgcgggcgtc atcattgacc gttttgagtt tatcccggtg
1920accgcgacct tcgaagcgga gtacgatctg gagcgtgcgc aggaa
196513655PRTArtificial SequenceCry1B variant 13Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Gln Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
141965DNAArtificial Sequencecoding sequence of Cry1B variant
14atgccgagca atcgtaagaa tgaaaatgaa atcattaacg cactgtccat ccctgcagtg
60agcaatcaca gcgcgcagat ggatttgagc ctggatgcgc gtatcgagga cagcctgtgt
120attgccgagg gcaacaacat caatccgttg gtcagcgcga gcaccgtgca
aaccggcatt 180aacattgccg gtcgtatcct gggtgtcctg ggcgttccgt
ttgcgggtca gctggcgagc 240ttttacagct ttatcgttgg tgagttgtgg
ccgtcgggtc gtgacccttg ggagattttc 300atggagcacg tcgagcaact
ggtgcgccaa cagattacgg agaatgcgcg caacaccgct 360ctggcgcgtc
tgcaaggtct gggtgcaagc ttccgcgctt accagcagtc cctggaagat
420tggttggaaa accgtgataa tgcgcgcact cgctccgtcc tgtacacgca
gtacatcgcg 480ctggagctgg acttcttgaa cgcgatgccg ctgtttgcaa
tcaacaacca gcaagtgccg 540ctgctgatgg tctacgccca agccgcgaat
ctgcacttgc tgctgctgcg cgacgcatct 600ctgttcggta gcgaatttgg
cctgaccagc caggagatcc agcgctacta tgagcgtcag 660gccgagaaaa
cgcgtgaata ctccgactac tgcgctcgtt ggtacaacac gggtctgaac
720aatctgcgtg gcaccaacgc ggagtcctgg ctgcgttaca accagtttcg
tcgcgatctg 780accctgggtg ttttggattt ggttgcgctg tttccgagct
atgacacccg catctatccg 840atcaacacca gcgcgcaact gactcgtgaa
atctatacgg acccgattgg ccgcactaat 900gcaccgtccg gtttcgcaag
caccaactgg ttcaataaca atgcaccgag cttcagcgcg 960atcgaggccg
cgatctttcg tccgccgcac ctgttggact tcccggagca gctgaccatc
1020tactctgcat ctagccgttg gagcagcacg cagcacatga attactgggt
tggccatcgt 1080ctgaacttcc gcccgattgg tggtacgctg aacactagca
cgcacggtgc cactaacacg 1140agcatcaacc cggtgacgct gcaattcacc
agccgtgatg tttaccgtac cgagtcctac 1200gccggcatca acattctgct
gaccaccccg gttaacggcg tcccttgggc tcgtttcaat 1260tggcgtaacc
cactgaatag cctgcgtggt tctttgctgt acaccattgg ttataccggc
1320gtcggtacgc aactgtttga ctcggaaact gagctgccac cggaaactac
cgagcgtccg 1380aactacgaat cttatagcca ccgtctgtcc aatatccgtc
tgatcatcgg caacaccctg 1440cgtgcgccgg tgtacagctg gacccatcgt
agcgccgatc gcacgaacac gattgccacc 1500aacattatca cccagatccc
ggcagtgaaa ggcaactttc tgtttaacgg cagcgtgatc 1560agcggtccag
gttttaccgg cggtgacctg gtgcgcctga acaacagcgg caacaatatc
1620caaaaccgtg gttatatcga agtcccgatt caattcatca gcacgagcac
ccgttaccgc 1680gtccgtgttc gctacgcatc cgttacgccg atccgcctga
gcgttaactg gggcaattcc 1740aacattttca gcagcattgt ccctgctacg
gcgacctctc tggacaattt gcagagccgt 1800aacttcggct atttcgaaag
ccgcaacgct ttcaccagcg ctacgggcaa tgtggttggt 1860gttcgcaatt
tcagcgagaa tgcgggcgtc atcattgacc gttttgagtt tatcccggtg
1920accgcgacct tcgaagcgga gtacgatctg gagcgtgcgc aggaa
196515655PRTArtificial SequenceCry1B variant 15Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Gln Ile 100 105 110 Thr Glu
Asn Ala Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125
Ala Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130
135 140 Arg Asp Asn Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile
Ala 145 150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe
Ala Ile Asn Asn 165 170 175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser
Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln
Arg Tyr Tyr Glu Arg Gln Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser
Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn
Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250
255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro
260 265 270 Ser Tyr Asp Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln
Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn
Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn
Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Ile Phe Arg
Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr
Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met Asn Tyr
Trp Val Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr
Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375
380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr
385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly
Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser
Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val
Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu
Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu
Ser Asn Ile Arg Leu Ile Ile Ser Asn Thr Leu 465 470 475 480 Arg Ala
Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495
Thr Ile Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500
505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly
Gly 515 520 525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln
Asn Arg Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr
Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val
Thr Pro Ile Arg Leu Ser Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile
Phe Ser Ser Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn
Leu Gln Ser Arg Asn Phe Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala
Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe 610 615 620
Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625
630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln
Glu 645 650 655 161965DNAArtificial Sequencecoding sequence of
Cry1B variant 16atgccgagca atcgtaagaa tgaaaatgaa atcattaacg
cactgtccat ccctgcagtg 60agcaatcaca gcgcgcagat ggatttgagc ctggatgcgc
gtatcgagga cagcctgtgt 120attgccgagg gcaacaacat caatccgttg
gtcagcgcga gcaccgtgca aaccggcatt 180aacattgccg gtcgtatcct
gggtgtcctg ggcgttccgt ttgcgggtca gctggcgagc 240ttttacagct
ttatcgttgg tgagttgtgg ccgtcgggtc gtgacccttg ggagattttc
300atggagcacg tcgagcaact ggtgcgccaa cagattacgg agaatgcgcg
caacaccgct 360ctggcgcgtc tgcaaggtct gggtgcaagc ttccgcgctt
accagcagtc cctggaagat 420tggttggaaa accgtgataa tgcgcgcact
cgctccgtcc tgtacacgca gtacatcgcg 480ctggagctgg acttcttgaa
cgcgatgccg ctgtttgcaa tcaacaacca gcaagtgccg 540ctgctgatgg
tctacgccca agccgcgaat ctgcacttgc tgctgctgcg cgacgcatct
600ctgttcggta gcgaatttgg cctgaccagc caggagatcc agcgctacta
tgagcgtcag 660gccgagaaaa cgcgtgaata ctccgactac tgcgctcgtt
ggtacaacac gggtctgaac 720aatctgcgtg gcaccaacgc ggagtcctgg
ctgcgttaca accagtttcg tcgcgatctg 780accctgggtg ttttggattt
ggttgcgctg tttccgagct atgacacccg catctatccg 840atcaacacca
gcgcgcaact gactcgtgaa atctatacgg acccgattgg ccgcactaat
900gcaccgtccg gtttcgcaag caccaactgg ttcaataaca atgcaccgag
cttcagcgcg 960atcgaggccg cgatctttcg tccgccgcac ctgttggact
tcccggagca gctgaccatc 1020tactctgcat ctagccgttg gagcagcacg
cagcacatga attactgggt tggccatcgt 1080ctgaacttcc gcccgattgg
tggtacgctg aacactagca cgcacggtgc cactaacacg 1140agcatcaacc
cggtgacgct gcaattcacc agccgtgatg tttaccgtac cgagtcctac
1200gccggcatca acattctgct gaccaccccg gttaacggcg tcccttgggc
tcgtttcaat 1260tggcgtaacc cactgaatag cctgcgtggt tctttgctgt
acaccattgg ttataccggc 1320gtcggtacgc aactgtttga ctcggaaact
gagctgccac cggaaactac cgagcgtccg 1380aactacgaat cttatagcca
ccgtctgtcc aatatccgtc tgatcatcag caacaccctg 1440cgtgcgccgg
tgtacagctg gacccatcgt agcgccgatc gcacgaacac gattgccacc
1500aacattatca cccagatccc ggcagtgaaa ggcaactttc tgtttaacgg
cagcgtgatc 1560agcggtccag gttttaccgg cggtgacctg gtgcgcctga
acaacagcgg caacaatatc 1620caaaaccgtg gttatatcga agtcccgatt
caattcatca gcacgagcac ccgttaccgc 1680gtccgtgttc gctacgcatc
cgttacgccg atccgcctga gcgttaactg gggcaattcc 1740aacattttca
gcagcattgt ccctgctacg gcgacctctc tggacaattt gcagagccgt
1800aacttcggct atttcgaaag ccgcaacgct ttcaccagcg ctacgggcaa
tgtggttggt 1860gttcgcaatt tcagcgagaa tgcgggcgtc atcattgacc
gttttgagtt tatcccggtg 1920accgcgacct tcgaagcgga gtacgatctg
gagcgtgcgc aggaa 196517655PRTArtificial SequenceCry1B variant 17Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10
15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp
20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn
Ile Asn 35 40 45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe
Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu
Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu
His Val Glu Gln Leu Val Arg Gln Ala Ile 100 105 110 Thr Leu Asn Ala
Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser
Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140
Arg Asp Asn Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145
150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile
Asn Asn 165 170 175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala
Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr
Tyr Glu Arg Gln Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr
Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg
Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg
Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265
270 Ser Tyr Asp Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr
275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro
Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro
Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro
His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala
Ser Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val
Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn
Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val
Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390
395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro
Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg
Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr
Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn
Ile Arg Leu Ile Ile Ser Gly Thr Leu 465 470 475 480 Arg Ala Pro Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile
Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515
520 525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg
Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr
Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro
Ile Arg Leu Ser Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser
Ser Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln
Ser Arg Asn Phe Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr
Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu
Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635
640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645
650 655 181965DNAArtificial Sequencecoding sequence of Cry1B
variant 18atgccgagca atcgtaagaa tgaaaatgaa atcattaacg cactgtccat
ccctgcagtg 60agcaatcaca gcgcgcagat ggatttgagc ctggatgcgc gtatcgagga
cagcctgtgt 120attgccgagg gcaacaacat caatccgttg gtcagcgcga
gcaccgtgca aaccggcatt 180aacattgccg gtcgtatcct gggtgtcctg
ggcgttccgt ttgcgggtca gctggcgagc 240ttttacagct ttatcgttgg
tgagttgtgg ccgtcgggtc gtgacccttg ggagattttc 300atggagcacg
tcgagcaact ggtgcgccaa gcgattacgc tgaatgcgcg caacaccgct
360ctggcgcgtc tgcaaggtct gggtgcaagc ttccgcgctt accagcagtc
cctggaagat 420tggttggaaa accgtgataa tgcgcgcact cgctccgtcc
tgtacacgca gtacatcgcg 480ctggagctgg acttcttgaa cgcgatgccg
ctgtttgcaa tcaacaacca gcaagtgccg 540ctgctgatgg tctacgccca
agccgcgaat ctgcacttgc tgctgctgcg cgacgcatct 600ctgttcggta
gcgaatttgg cctgaccagc caggagatcc agcgctacta tgagcgtcag
660gccgagaaaa cgcgtgaata ctccgactac tgcgctcgtt ggtacaacac
gggtctgaac 720aatctgcgtg gcaccaacgc ggagtcctgg ctgcgttaca
accagtttcg tcgcgatctg 780accctgggtg ttttggattt ggttgcgctg
tttccgagct atgacacccg catctatccg 840atcaacacca gcgcgcaact
gactcgtgaa atctatacgg acccgattgg ccgcactaat 900gcaccgtccg
gtttcgcaag caccaactgg ttcaataaca atgcaccgag cttcagcgcg
960atcgaggccg cgatctttcg tccgccgcac ctgttggact tcccggagca
gctgaccatc 1020tactctgcat ctagccgttg gagcagcacg cagcacatga
attactgggt tggccatcgt 1080ctgaacttcc gcccgattgg tggtacgctg
aacactagca cgcacggtgc cactaacacg 1140agcatcaacc cggtgacgct
gcaattcacc agccgtgatg tttaccgtac cgagtcctac 1200gccggcatca
acattctgct gaccaccccg gttaacggcg tcccttgggc tcgtttcaat
1260tggcgtaacc cactgaatag cctgcgtggt tctttgctgt acaccattgg
ttataccggc 1320gtcggtacgc aactgtttga ctcggaaact gagctgccac
cggaaactac cgagcgtccg 1380aactacgaat cttatagcca ccgtctgtcc
aatatccgtc tgatcatcag cggcaccctg 1440cgtgcgccgg tgtacagctg
gacccatcgt agcgccgatc gcacgaacac gattgccacc 1500aacattatca
cccagatccc ggcagtgaaa ggcaactttc tgtttaacgg cagcgtgatc
1560agcggtccag gttttaccgg cggtgacctg gtgcgcctga acaacagcgg
caacaatatc 1620caaaaccgtg gttatatcga agtcccgatt caattcatca
gcacgagcac ccgttaccgc 1680gtccgtgttc gctacgcatc cgttacgccg
atccgcctga gcgttaactg gggcaattcc 1740aacattttca gcagcattgt
ccctgctacg gcgacctctc tggacaattt gcagagccgt 1800aacttcggct
atttcgaaag ccgcaacgct ttcaccagcg ctacgggcaa tgtggttggt
1860gttcgcaatt tcagcgagaa tgcgggcgtc atcattgacc gttttgagtt
tatcccggtg 1920accgcgacct tcgaagcgga gtacgatctg gagcgtgcgc aggaa
196519655PRTArtificial SequenceCry1B variant 19Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Ala Ile 100 105 110 Thr Leu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Tyr Phe Arg Pro Ile Asn Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Asn Thr Leu 465
470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg
Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala
Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly
Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu Val Arg Leu Asn Asn Ser
Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540 Tyr Ile Glu Val Pro Ile
Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val
Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser Val Asn 565 570 575 Trp
Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro Ala Thr Ala Thr 580 585
590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe Gly Tyr Phe Glu Ser Arg
595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg
Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu
Phe Ile Pro Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp
Leu Glu Arg Ala Gln Glu 645 650 655 201965DNAArtificial
Sequencecoding sequence of Cry1B variant 20atgccgagca atcgtaagaa
tgaaaatgaa atcattaacg cactgtccat ccctgcagtg 60agcaatcaca gcgcgcagat
ggatttgagc ctggatgcgc gtatcgagga cagcctgtgt 120attgccgagg
gcaacaacat caatccgttg gtcagcgcga gcaccgtgca aaccggcatt
180aacattgccg gtcgtatcct gggtgtcctg ggcgttccgt ttgcgggtca
gctggcgagc 240ttttacagct ttatcgttgg tgagttgtgg ccgtcgggtc
gtgacccttg ggagattttc 300atggagcacg tcgagcaact ggtgcgccaa
gcgattacgc tgaatgcgcg caacaccgct 360ctggcgcgtc tgcaaggtct
gggtgcaagc ttccgcgctt accagcagtc cctggaagat 420tggttggaaa
accgtgataa tgcgcgcact cgctccgtcc tgtacacgca gtacatcgcg
480ctggagctgg acttcttgaa cgcgatgccg ctgtttgcaa tcaacaacca
gcaagtgccg 540ctgctgatgg tctacgccca agccgcgaat ctgcacttgc
tgctgctgcg cgacgcatct 600ctgttcggta gcgaatttgg cctgaccagc
caggagatcc agcgctacta tgagcgtcag 660gccgagaaaa cgcgtgaata
ctccgactac tgcgctcgtt ggtacaacac gggtctgaac 720aatctgcgtg
gcaccaacgc ggagtcctgg ctgcgttaca accagtttcg tcgcgatctg
780accctgggtg ttttggattt ggttgcgctg tttccgagct atgacacccg
catctatccg 840atcaacacca gcgcgcaact gactcgtgaa atctatacgg
acccgattgg ccgcactaat 900gcaccgtccg gtttcgcaag caccaactgg
ttcaataaca atgcaccgag cttcagcgcg 960atcgaggccg cgatctttcg
tccgccgcac ctgttggact tcccggagca gctgaccatc 1020tactctgcat
ctagccgttg gagcagcacg cagcacatga attactgggt tggccatcgt
1080ctgtatttcc gcccgattaa cggtacgctg aacactagca cgcacggtgc
cactaacacg 1140agcatcaacc cggtgacgct gcaattcacc agccgtgatg
tttaccgtac cgagtcctac 1200gccggcatca acattctgct gaccaccccg
gttaacggcg tcccttgggc tcgtttcaat 1260tggcgtaacc cactgaatag
cctgcgtggt tctttgctgt acaccattgg ttataccggc 1320gtcggtacgc
aactgtttga ctcggaaact gagctgccac cggaaactac cgagcgtccg
1380aactacgaat cttatagcca ccgtctgtcc aatatccgtc tgatcatcgg
caacaccctg 1440cgtgcgccgg tgtacagctg gacccatcgt agcgccgatc
gcacgaacac gattgccacc 1500aacattatca cccagatccc ggcagtgaaa
ggcaactttc tgtttaacgg cagcgtgatc 1560agcggtccag gttttaccgg
cggtgacctg gtgcgcctga acaacagcgg caacaatatc 1620caaaaccgtg
gttatatcga agtcccgatt caattcatca gcacgagcac ccgttaccgc
1680gtccgtgttc gctacgcatc cgttacgccg atccgcctga gcgttaactg
gggcaattcc 1740aacattttca gcagcattgt ccctgctacg gcgacctctc
tggacaattt gcagagccgt 1800aacttcggct atttcgaaag ccgcaacgct
ttcaccagcg ctacgggcaa tgtggttggt 1860gttcgcaatt tcagcgagaa
tgcgggcgtc atcattgacc gttttgagtt tatcccggtg 1920accgcgacct
tcgaagcgga gtacgatctg gagcgtgcgc aggaa 196521655PRTArtificial
SequenceCry1B variant 21Met Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile
Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn His Ser Ala
Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala Arg Ile Glu Asp Ser Leu
Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40 45 Pro Leu Val Ser Ala
Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60 Arg Ile Leu
Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe
Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro 85 90
95 Trp Glu Ile Phe Met Glu His Val Glu Gln Leu Val Arg Gln Ala Ile
100 105 110 Thr Leu Asn Ala Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly
Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp
Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala Arg Thr Arg Ser Val Leu
Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu Glu Leu Asp Phe Leu Asn
Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170 175 Gln Gln Val Pro Leu
Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185 190 Leu Leu Leu
Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu 195 200 205 Thr
Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Ala Glu Lys Thr 210 215
220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn
225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr
Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu
Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg Ile Tyr Pro Ile
Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile Tyr Thr Asp Pro
Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe Ala Ser Thr Asn
Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310 315 320 Ile Glu
Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro Glu 325 330 335
Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His 340
345 350 Met Asn Tyr Trp Val Gly His Arg Leu Tyr Phe Arg Pro Ile Gln
Gly 355 360 365 Thr Leu Asn Thr Ser Thr His Gly Ala Thr Asn Thr Ser
Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr
Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly Ile Asn Ile Leu Leu Thr
Thr Pro Val Asn Gly Val Pro Trp 405 410 415 Ala Arg Phe Asn Trp Arg
Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430 Leu Tyr Thr Ile
Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435 440 445 Glu Thr
Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser 450 455 460
Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ile Gly Asn Thr Leu 465
470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg
Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala
Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn Gly Ser Val Ile Ser Gly
Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu Val Arg Leu Asn Asn Ser
Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540 Tyr Ile Glu Val Pro Ile
Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545 550 555 560 Val Arg Val
Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser Val Asn 565 570 575 Trp
Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro Ala Thr Ala Thr 580 585
590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe Gly Tyr Phe Glu Ser Arg
595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg
Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu
Phe Ile Pro Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp
Leu Glu Arg Ala Gln Glu 645 650 655 221965DNAArtificial
Sequencecoding sequence of Cry1B variant 22atgccgagca atcgtaagaa
tgaaaatgaa atcattaacg cactgtccat ccctgcagtg 60agcaatcaca gcgcgcagat
ggatttgagc ctggatgcgc gtatcgagga cagcctgtgt 120attgccgagg
gcaacaacat caatccgttg gtcagcgcga gcaccgtgca aaccggcatt
180aacattgccg gtcgtatcct gggtgtcctg ggcgttccgt ttgcgggtca
gctggcgagc 240ttttacagct ttatcgttgg tgagttgtgg ccgtcgggtc
gtgacccttg ggagattttc 300atggagcacg tcgagcaact ggtgcgccaa
gcgattacgc tgaatgcgcg caacaccgct 360ctggcgcgtc tgcaaggtct
gggtgcaagc ttccgcgctt accagcagtc cctggaagat 420tggttggaaa
accgtgataa tgcgcgcact cgctccgtcc tgtacacgca gtacatcgcg
480ctggagctgg acttcttgaa cgcgatgccg ctgtttgcaa tcaacaacca
gcaagtgccg 540ctgctgatgg tctacgccca agccgcgaat ctgcacttgc
tgctgctgcg cgacgcatct 600ctgttcggta gcgaatttgg cctgaccagc
caggagatcc agcgctacta tgagcgtcag 660gccgagaaaa cgcgtgaata
ctccgactac tgcgctcgtt ggtacaacac gggtctgaac 720aatctgcgtg
gcaccaacgc ggagtcctgg ctgcgttaca accagtttcg tcgcgatctg
780accctgggtg ttttggattt ggttgcgctg tttccgagct atgacacccg
catctatccg 840atcaacacca gcgcgcaact gactcgtgaa atctatacgg
acccgattgg ccgcactaat 900gcaccgtccg gtttcgcaag caccaactgg
ttcaataaca atgcaccgag cttcagcgcg 960atcgaggccg cgatctttcg
tccgccgcac ctgttggact tcccggagca gctgaccatc 1020tactctgcat
ctagccgttg gagcagcacg cagcacatga attactgggt tggccatcgt
1080ctgtatttcc gcccgattca gggtacgctg aacactagca cgcacggtgc
cactaacacg 1140agcatcaacc cggtgacgct gcaattcacc agccgtgatg
tttaccgtac cgagtcctac 1200gccggcatca acattctgct gaccaccccg
gttaacggcg tcccttgggc tcgtttcaat 1260tggcgtaacc cactgaatag
cctgcgtggt tctttgctgt acaccattgg ttataccggc 1320gtcggtacgc
aactgtttga ctcggaaact gagctgccac cggaaactac cgagcgtccg
1380aactacgaat cttatagcca ccgtctgtcc aatatccgtc tgatcatcgg
caacaccctg 1440cgtgcgccgg tgtacagctg gacccatcgt agcgccgatc
gcacgaacac gattgccacc 1500aacattatca cccagatccc ggcagtgaaa
ggcaactttc tgtttaacgg cagcgtgatc 1560agcggtccag gttttaccgg
cggtgacctg gtgcgcctga acaacagcgg caacaatatc 1620caaaaccgtg
gttatatcga agtcccgatt caattcatca gcacgagcac ccgttaccgc
1680gtccgtgttc gctacgcatc cgttacgccg atccgcctga gcgttaactg
gggcaattcc 1740aacattttca gcagcattgt ccctgctacg gcgacctctc
tggacaattt gcagagccgt 1800aacttcggct atttcgaaag ccgcaacgct
ttcaccagcg ctacgggcaa tgtggttggt 1860gttcgcaatt tcagcgagaa
tgcgggcgtc atcattgacc gttttgagtt tatcccggtg 1920accgcgacct
tcgaagcgga gtacgatctg gagcgtgcgc aggaa 196523663PRTArtificial
SequenceCry1B variant 23Met Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile
Ile Asn Ala Val Ser 1 5 10 15 Asn His Ser Ala Gln Met Asp Leu Ser
Leu Asp Ala Arg Ile Glu Asp 20 25 30 Ser Leu Cys Val Ala Glu Val
Asn Asn Ile Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val Gln Thr
Gly Ile Ser Ile Ala Gly Arg Ile Leu Gly Val 50 55 60 Leu Gly Val
Pro Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val
Gly Glu Leu Trp Pro Ser Gly Arg Asp Pro Trp Glu Ile Phe Met 85 90
95 Glu His Val Glu Gln Ile Val Arg Gln Gln Ile Thr Asp Ser Val Arg
100 105 110 Asp Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly Arg Gly Tyr
Arg Ser 115 120 125 Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg
Asn Asp Ala Arg 130 135 140 Ser Arg Ser Ile Ile Arg Glu Arg Tyr Ile
Ala Leu Glu Leu Asp Ile 145 150 155 160 Thr Thr Ala Ile Pro Leu Phe
Ser Ile Arg Asn Gln Glu Val Pro Leu 165 170 175 Leu Met Val Tyr Ala
Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190 Asp Ala Ser
Leu Phe Gly Ser Glu Trp Gly Met Ser Ser Ser Asp Val 195 200 205 Asn
Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr Thr Glu Glu Tyr Ser Asn 210 215
220 His Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr
225 230 235 240 Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg
Asp Leu Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro
Ser Tyr Asp Thr Arg 260 265 270 Val Tyr Pro Met Asn Thr Ser Ala Gln
Leu Thr Arg Glu Ile Tyr Thr 275 280 285 Asp Pro Ile Gly Arg Thr Asn
Ala Pro Ser Gly Phe Ala Ser Thr Asn 290 295 300 Trp Phe Asn Asn Asn
Ala Pro Ser Phe Ser Ala Ile Glu Ala Ala Ile 305 310 315 320 Phe Arg
Pro Pro His Leu Leu Asp Phe Pro Glu Gln Leu Thr Ile Tyr 325 330 335
Ser Ala Ser Ser Arg Trp Ser Ser Thr Gln His Met Asn Tyr Trp Val 340
345 350 Gly His Arg Leu Asn Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr
Ser 355 360 365 Thr Gln Gly Leu Thr Asn Asn Thr Ser Ile Asn Pro Val
Thr Leu Gln 370 375 380 Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser
Asn Ala Gly Thr Asn 385 390 395 400 Ile Leu Phe Thr Thr Pro Val Asn
Gly Val Pro Trp Ala Arg Phe Asn 405 410 415 Phe Ile Asn Pro Gln Asn
Ile Tyr Glu Arg Gly Ala Thr Thr Tyr Ser 420 425 430 Gln Pro Tyr Gln
Gly Val Gly Ile Gln Leu Phe Asp Ser Glu Thr Glu 435 440 445 Leu Pro
Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His 450 455 460
Arg Leu Ser His Ile Gly Leu Ile Ile Gly Asn Thr Leu Arg Ala Pro 465
470 475 480 Val Tyr Ser Trp Thr His Arg Ser Ala Thr Leu Thr Asn Thr
Ile Asp 485 490 495 Pro Glu Arg Ile Asn Gln Ile Pro Leu Val Lys Gly
Phe Arg Val Trp 500 505 510 Gly Gly Thr Ser Val Ile Thr Gly Pro Gly
Phe Thr Gly Gly Asp Ile 515 520 525 Leu Arg Arg Asn Thr Phe Gly Asp
Phe Val Ser Leu Gln Val Asn Ile 530 535 540 Asn Ser Pro Ile Thr Gln
Arg Tyr Arg Leu Arg Phe Arg Tyr Ala Ser 545 550 555 560 Ser Arg Asp
Ala Arg Val Ile Val Leu Thr Gly Ala Ala Ser Thr Gly 565 570 575 Val
Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys Thr Met Glu 580 585
590 Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser
595 600 605 Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile
Ser Glu 610 615 620 Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly
Glu Leu Tyr Ile 625 630 635 640 Asp Lys Ile Glu Ile Ile Leu Ala Asp
Ala Thr Phe Glu Ala Glu Ser 645 650 655 Asp Leu Glu Arg Ala Gln Lys
660 241989DNAArtificial Sequencecoding sequence of Cry1B variant
24atgccttcaa ataggaaaaa tgagaatgaa attataaatg ctgtatcgaa tcattccgca
60caaatggatc tatcgctaga tgctcgtatt gaagatagct tgtgtgtagc cgaggtgaac
120aatattgatc catttgttag cgcatcaaca gtccaaacag gtattagtat
agctggtaga 180atattgggcg tattaggtgt gccgtttgct ggacaactag
ctagttttta tagttttctt 240gttggggaat tatggcctag cggcagagat
ccatgggaaa tttttatgga acatgtcgag 300caaattgtaa gacaacaaat
aacggacagt gttagggata ccgctattgc tcgtttagaa 360ggtctaggaa
gagggtatag atcttaccag caggctcttg aaacttggtt agataaccga
420aatgatgcaa gatcaagaag cattattcgt gagagatata ttgctttaga
acttgacatt 480actactgcta taccgctttt cagcatacga aatcaagagg
ttccattatt aatggtatat 540gctcaagctg caaatttaca cctattatta
ttgagagacg catccctttt tggtagtgaa 600tgggggatgt catcttccga
tgttaaccaa tattaccaag aacaaatcag atatacagag 660gaatattcta
accattgcgt acaatggtat aatacagggc taaataactt aagagggaca
720aatgctgaaa gttggttgcg gtataatcaa ttccgtagag atctaacgtt
aggagtatta 780gatctagtgg cactattccc aagctatgac acgcgtgttt
atccaatgaa tacgagtgct 840cagttaacaa gagaaattta tacagatcca
attgggagaa caaatgcacc ttcaggattt 900gcaagtacga attggtttaa
taataatgca ccatcgtttt ctgccataga ggctgccatt 960ttcaggcctc
cgcatctact tgattttcca gaacaactta caatttacag tgcatcaagc
1020cgttggagta
gcactcaaca tatgaattat tgggtgggac ataggcttaa cttccgccca
1080ataggaggga cattaaatac ctcaacacaa ggacttacta ataatacttc
aattaatcct 1140gtaacattac agtttacgtc tcgtgacgtt tatagaacag
aatcaaatgc agggacaaat 1200atactattta ctactcctgt gaatggagta
ccttgggcta gatttaattt tataaaccct 1260cagaatattt atgaaagagg
cgccactacc tacagtcaac cgtatcaggg agttgggatt 1320caattatttg
attcagaaac tgaattacca ccagaaacaa cagaacgacc aaattatgaa
1380tcatatagtc atagattatc tcatatagga ctaatcatag gaaacacttt
gagagcacca 1440gtctattctt ggacgcatcg tagtgcaact cttacaaata
caattgatcc agagagaatt 1500aatcaaatac ctttagtgaa aggatttaga
gtttgggggg gcacctctgt cattacagga 1560ccaggattta caggagggga
tatccttcga agaaatacct ttggtgattt tgtatctcta 1620caagtcaata
ttaattcacc aattacccaa agataccgtt taagatttcg ttacgcttcc
1680agtagggatg cacgagttat agtattaaca ggagcggcat ccacaggagt
gggaggccaa 1740gttagtgtaa atatgcctct tcagaaaact atggaaatag
gggagaactt aacatctaga 1800acatttagat ataccgattt tagtaatcct
ttttcattta gagctaatcc agatataatt 1860gggataagtg aacaacctct
atttggtgca ggttctatta gtagcggtga actttatata 1920gataaaattg
aaattattct agcagatgca acatttgaag cagaatctga tttagaaaga
1980gcacaaaag 198925663PRTArtificial SequenceCry1B variant 25Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser 1 5 10
15 Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp Ala Arg Ile Glu Asp
20 25 30 Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp Pro Phe Val
Ser Ala 35 40 45 Ser Thr Val Gln Thr Gly Ile Ser Ile Ala Gly Arg
Ile Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gln Leu Ala
Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Ser Gly
Arg Asp Pro Trp Glu Ile Phe Met 85 90 95 Glu His Val Glu Gln Ile
Val Arg Gln Gln Ile Thr Asp Ser Val Arg 100 105 110 Asp Thr Ala Ile
Ala Arg Leu Glu Gly Leu Gly Arg Gly Tyr Arg Ser 115 120 125 Tyr Gln
Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg Asn Asp Ala Arg 130 135 140
Ser Arg Ser Ile Ile Arg Glu Arg Tyr Ile Ala Leu Glu Leu Asp Ile 145
150 155 160 Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg Asn Gln Glu Val
Pro Leu 165 170 175 Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu
Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly
Met Ser Ser Ser Asp Val 195 200 205 Asn Gln Tyr Tyr Gln Glu Gln Ile
Arg Tyr Thr Glu Glu Tyr Ser Asn 210 215 220 His Cys Val Gln Trp Tyr
Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr 225 230 235 240 Asn Ala Glu
Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr 245 250 255 Leu
Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265
270 Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Ile Tyr Thr
275 280 285 Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly Phe Ala Ser
Thr Asn 290 295 300 Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile
Glu Ala Ala Ile 305 310 315 320 Phe Arg Pro Pro His Leu Leu Asp Phe
Pro Glu Gln Leu Thr Ile Tyr 325 330 335 Ser Ala Ser Ser Arg Trp Ser
Ser Thr Gln His Met Asn Tyr Trp Val 340 345 350 Gly His Arg Leu Asn
Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365 Thr Gln Gly
Leu Thr Asn Asn Thr Ser Ile Asn Pro Val Thr Leu Gln 370 375 380 Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Asn Ala Gly Thr Asn 385 390
395 400 Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe
Asn 405 410 415 Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly Ala Thr
Thr Tyr Ser 420 425 430 Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe
Asp Ser Glu Thr Glu 435 440 445 Leu Pro Pro Glu Thr Thr Glu Arg Pro
Asn Tyr Glu Ser Tyr Ser His 450 455 460 Arg Leu Ser His Ile Gly Leu
Ile Ile Gly Asn Thr Leu Arg Ala Pro 465 470 475 480 Val Tyr Ser Trp
Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 485 490 495 Pro Glu
Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 500 505 510
Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 515
520 525 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn
Ile 530 535 540 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
Tyr Ala Ser 545 550 555 560 Ser Arg Asp Ala Arg Val Ile Val Leu Thr
Gly Ala Ala Ser Thr Gly 565 570 575 Val Gly Gly Gln Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu 580 585 590 Ile Gly Glu Asn Leu Thr
Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 595 600 605 Asn Pro Phe Ser
Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 610 615 620 Gln Pro
Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 625 630 635
640 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser
645 650 655 Asp Leu Glu Gly Ala Arg Lys 660 261989DNAArtificial
Sequencecoding sequence of Cry1B variant 26atgccttcaa ataggaaaaa
tgagaatgaa attataaatg ctgtatcgaa tcattccgca 60caaatggatc tatcgctaga
tgctcgtatt gaagatagct tgtgtgtagc cgaggtgaac 120aatattgatc
catttgttag cgcatcaaca gtccaaacag gtattagtat agctggtaga
180atattgggcg tattaggtgt gccgtttgct ggacaactag ctagttttta
tagttttctt 240gttggggaat tatggcctag cggcagagat ccatgggaaa
tttttatgga acatgtcgag 300caaattgtaa gacaacaaat aacggacagt
gttagggata ccgctattgc tcgtttagaa 360ggtctaggaa gagggtatag
atcttaccag caggctcttg aaacttggtt agataaccga 420aatgatgcaa
gatcaagaag cattattcgt gagagatata ttgctttaga acttgacatt
480actactgcta taccgctttt cagcatacga aatcaagagg ttccattatt
aatggtatat 540gctcaagctg caaatttaca cctattatta ttgagagacg
catccctttt tggtagtgaa 600tgggggatgt catcttccga tgttaaccaa
tattaccaag aacaaatcag atatacagag 660gaatattcta accattgcgt
acaatggtat aatacagggc taaataactt aagagggaca 720aatgctgaaa
gttggttgcg gtataatcaa ttccgtagag atctaacgtt aggagtatta
780gatctagtgg cactattccc aagctatgac actcgcactt atccaatcaa
tacgagtgct 840cagttaacaa gagaaattta tacagatcca attgggagaa
caaatgcacc ttcaggattt 900gcaagtacga attggtttaa taataatgca
ccatcgtttt ctgccataga ggctgccatt 960ttcaggcctc cgcatctact
tgattttcca gaacaactta caatttacag tgcatcaagc 1020cgttggagta
gcactcaaca tatgaattat tgggtgggac ataggcttaa cttccgccca
1080ataggaggga cattaaatac ctcaacacaa ggacttacta ataatacttc
aattaatcct 1140gtaacattac agtttacgtc tcgtgacgtt tatagaacag
aatcaaatgc agggacaaat 1200atactattta ctactcctgt gaatggagta
ccttgggcta gatttaattt tataaaccct 1260cagaatattt atgaaagagg
cgccactacc tacagtcaac cgtatcaggg agttgggatt 1320caattatttg
attcagaaac tgaattacca ccagaaacaa cagaacgacc aaattatgaa
1380tcatatagtc atagattatc tcatatagga ctaatcatag gaaacacttt
gagagcacca 1440gtctattctt ggacgcatcg tagtgcaact cttacaaata
caattgatcc agagagaatt 1500aatcaaatac ctttagtgaa aggatttaga
gtttgggggg gcacctctgt cattacagga 1560ccaggattta caggagggga
tatccttcga agaaatacct ttggtgattt tgtatctcta 1620caagtcaata
ttaattcacc aattacccaa agataccgtt taagatttcg ttacgcttcc
1680agtagggatg cacgagttat agtattaaca ggagcggcat ccacaggagt
gggaggccaa 1740gttagtgtaa atatgcctct tcagaaaact atggaaatag
gggagaactt aacatctaga 1800acatttagat ataccgattt tagtaatcct
ttttcattta gagctaatcc agatataatt 1860gggataagtg aacaacctct
atttggtgca ggttctatta gtagcggtga actttatata 1920gataaaattg
aaattattct agcagatgca acatttgaag cagaatctga tttagaaggg
1980gcgcggaag 198927663PRTArtificial SequenceCry1B variant 27Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser 1 5 10
15 Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp Ala Arg Ile Glu Asp
20 25 30 Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp Pro Phe Val
Ser Ala 35 40 45 Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly Arg
Ile Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gln Leu Ala
Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Ser Gly
Arg Asp Pro Trp Glu Ile Phe Met 85 90 95 Glu His Val Glu Gln Ile
Val Arg Gln Gln Ile Thr Asp Ser Val Arg 100 105 110 Asp Thr Ala Ile
Ala Arg Leu Glu Gly Leu Gly Arg Gly Tyr Arg Ser 115 120 125 Tyr Gln
Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg Asn Asp Ala Arg 130 135 140
Ser Arg Ser Ile Ile Leu Glu Arg Tyr Ile Ala Leu Glu Leu Asp Ile 145
150 155 160 Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg Asn Gln Glu Val
Pro Leu 165 170 175 Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu
Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly
Met Ala Ser Ser Asp Val 195 200 205 Asn Gln Tyr Tyr Gln Glu Gln Ile
Arg Tyr Thr Glu Glu Tyr Ser Asn 210 215 220 His Cys Val Gln Trp Tyr
Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr 225 230 235 240 Asn Ala Glu
Ser Trp Leu Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr 245 250 255 Leu
Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265
270 Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Ile Tyr Thr
275 280 285 Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly Phe Ala Ser
Thr Asn 290 295 300 Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile
Glu Ala Ala Ile 305 310 315 320 Phe Arg Pro Pro His Leu Leu Asp Phe
Pro Glu Gln Leu Thr Ile Tyr 325 330 335 Ser Ala Ser Ser Arg Trp Ser
Ser Thr Gln His Met Asn Tyr Trp Val 340 345 350 Gly His Arg Leu Asn
Phe Arg Pro Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365 Thr Gln Gly
Leu Thr Asn Asn Thr Ser Ile Asn Pro Val Thr Leu Gln 370 375 380 Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Asn Ala Gly Thr Asn 385 390
395 400 Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe
Asn 405 410 415 Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly Ala Thr
Thr Tyr Ser 420 425 430 Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe
Asp Ser Glu Thr Glu 435 440 445 Leu Pro Pro Glu Thr Thr Glu Arg Pro
Asn Tyr Glu Ser Tyr Ser His 450 455 460 Arg Leu Ser His Ile Gly Leu
Ile Ile Gly Asn Thr Leu Arg Ala Pro 465 470 475 480 Val Tyr Ser Trp
Thr His Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 485 490 495 Pro Glu
Arg Ile Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 500 505 510
Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 515
520 525 Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn
Ile 530 535 540 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
Tyr Ala Ser 545 550 555 560 Ser Arg Asp Ala Arg Val Ile Val Leu Thr
Gly Ala Ala Ser Thr Gly 565 570 575 Val Gly Gly Gln Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu 580 585 590 Ile Gly Glu Asn Leu Thr
Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser 595 600 605 Asn Pro Phe Ser
Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 610 615 620 Gln Pro
Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 625 630 635
640 Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser
645 650 655 Asp Leu Glu Lys Ala Gln Lys 660 281989DNAArtificial
Sequencecoding sequence of Cry1B variant 28atgccttcaa ataggaaaaa
tgagaatgaa attataaatg ctgtatcgaa tcattccgca 60caaatggatc tatcgctaga
tgctcgtatt gaagatagct tgtgtgtagc cgaggtgaac 120aatattgatc
catttgttag cgcatcaaca gtccaaacgg gtattaacat agctggtaga
180atactaggcg tattaggggt gccgtttgct ggacaactag ctagttttta
tagttttctt 240gttggggaat tatggcctag cggcagagat ccatgggaaa
tttttatgga acatgtcgag 300caaattgtaa gacaacaaat aacggacagt
gttagggata ccgctattgc tcgtttagaa 360ggtctaggaa gagggtatag
atcttaccag caggctcttg aaacttggtt agataaccga 420aatgatgcaa
gatcaagaag cattattctt gagcgctata ttgctttaga acttgacatt
480actactgcta taccgctttt cagcatacga aatcaagagg ttccattatt
gatggtatat 540gctcaagctg caaatttaca cctattatta ttgagagacg
catccctttt tggtagtgaa 600tgggggatgg catcttccga tgttaaccaa
tattaccaag aacaaatcag atatacagag 660gaatattcta accattgcgt
acaatggtat aatacagggc taaataactt aagagggaca 720aatgctgaaa
gttggttgcg gtataatcaa ttccgtagag acctaacgtt aggggtatta
780gatttagtag ccctattccc aagctatgat actcgcactt atccaatcaa
tacgagtgct 840cagttaacaa gagaaattta tacagatcca attgggagaa
caaatgcacc ttcaggattt 900gcaagtacga attggtttaa taataatgca
ccatcgtttt ctgccataga ggctgccatt 960ttcaggcctc cgcatctact
tgattttcca gaacaactta caatttacag tgcatcaagc 1020cgttggagta
gcactcaaca tatgaattat tgggtgggac ataggcttaa cttccgccca
1080ataggaggga cattaaatac ctcaacacaa ggacttacta ataatacttc
aattaatcct 1140gtaacattac agtttacgtc tcgtgacgtt tatagaacag
aatcaaatgc agggacaaat 1200atactattta ctactcctgt gaatggagta
ccttgggcta gatttaattt tataaaccct 1260cagaatattt atgaaagagg
cgccactacc tacagtcaac cgtatcaggg agttgggatt 1320caattatttg
attcagaaac tgaattacca ccagaaacaa cagaacgacc aaattatgaa
1380tcatatagtc atagattatc tcatatagga ctaatcatag gaaacacttt
gagagcacca 1440gtctattctt ggacgcatcg tagtgcaact cttacaaata
caattgatcc agagagaatt 1500aatcaaatac ctttagtgaa aggatttaga
gtttgggggg gcacctctgt cattacagga 1560ccaggattta caggagggga
tatccttcga agaaatacct ttggtgattt tgtatctcta 1620caagtcaata
ttaattcacc aattacccaa agataccgtt taagatttcg ttacgcttcc
1680agtagggatg cacgagttat agtattaaca ggagcggcat ccacaggagt
gggaggccaa 1740gttagtgtaa atatgcctct tcagaaaact atggaaatag
gggagaactt aacatctaga 1800acatttagat ataccgattt tagtaatcct
ttttcattta gagctaatcc agatataatt 1860gggataagtg aacaacctct
atttggtgca ggttctatta gtagcggtga actttatata 1920gataaaattg
aaattattct agcagatgca acatttgaag cagaatctga tttagagaaa
1980gctcagaaa 198929663PRTArtificial SequenceCry1B variant 29Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Val Ser 1 5 10
15 Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp Ala Arg Ile Glu Asp
20 25 30 Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp Pro Phe Val
Ser Ala 35 40 45 Ser Thr Val Gln Thr Gly Ile Ser Ile Ala Gly Arg
Ile Leu Gly Val 50 55 60 Leu Gly Val Pro Phe Ala Gly Gln Leu Ala
Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu Leu Trp Pro Ser Gly
Arg Asp Pro Trp Glu Ile Phe Leu 85 90 95 Glu His Val Glu Gln Leu
Ile Arg Gln Gln Val Thr Glu Asn Thr Arg 100 105 110 Asn Thr Ala Ile
Ala Arg Leu Glu Gly Leu Gly Arg Gly Tyr Arg Ser 115 120 125 Tyr Gln
Gln Ala Leu Glu Thr Trp Leu Asp Asn Arg Asn Asp Ala Arg 130 135 140
Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala Leu Glu Leu Asp Ile 145
150 155
160 Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg Asn Gln Glu Val Pro Leu
165 170 175 Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu Leu
Leu Arg 180 185 190 Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met Ser
Ser Ala Asp Val 195 200 205 Asn Gln Tyr Tyr Gln Glu Gln Ile Arg Tyr
Thr Glu Glu Tyr Ser Asn 210 215 220 His Cys Val Gln Trp Tyr Asn Thr
Gly Leu Asn Asn Leu Arg Gly Thr 225 230 235 240 Asn Ala Glu Ser Trp
Leu Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr 245 250 255 Leu Gly Val
Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg 260 265 270 Thr
Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr Arg Glu Ile Tyr Thr 275 280
285 Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly Phe Ala Ser Thr Asn
290 295 300 Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu Ala
Ala Ile 305 310 315 320 Phe Arg Pro Pro His Leu Leu Asp Phe Pro Glu
Gln Leu Thr Ile Tyr 325 330 335 Ser Ala Ser Ser Arg Trp Ser Ser Thr
Gln His Met Asn Tyr Trp Val 340 345 350 Gly His Arg Leu Asn Phe Arg
Pro Ile Gly Gly Thr Leu Asn Thr Ser 355 360 365 Thr Gln Gly Leu Thr
Asn Asn Thr Ser Ile Asn Pro Val Thr Leu Gln 370 375 380 Phe Thr Ser
Arg Asp Val Tyr Arg Thr Glu Ser Asn Ala Gly Thr Asn 385 390 395 400
Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro Trp Ala Arg Phe Asn 405
410 415 Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly Ala Thr Thr Tyr
Ser 420 425 430 Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe Asp Ser
Glu Thr Glu 435 440 445 Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr
Glu Ser Tyr Ser His 450 455 460 Arg Leu Ser His Ile Gly Leu Ile Ile
Gly Asn Thr Leu Arg Ala Pro 465 470 475 480 Val Tyr Ser Trp Thr His
Arg Ser Ala Thr Leu Thr Asn Thr Ile Asp 485 490 495 Pro Glu Arg Ile
Asn Gln Ile Pro Leu Val Lys Gly Phe Arg Val Trp 500 505 510 Gly Gly
Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly Gly Asp Ile 515 520 525
Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln Val Asn Ile 530
535 540 Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg Tyr Ala
Ser 545 550 555 560 Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala
Ala Ser Thr Gly 565 570 575 Val Gly Gly Gln Val Ser Val Asn Met Pro
Leu Gln Lys Thr Met Glu 580 585 590 Ile Gly Glu Asn Leu Thr Ser Arg
Thr Phe Arg Tyr Thr Asp Phe Ser 595 600 605 Asn Pro Phe Ser Phe Arg
Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu 610 615 620 Gln Pro Leu Phe
Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile 625 630 635 640 Asp
Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu Ala Glu Ser 645 650
655 Asp Leu Glu Arg Ala Gln Lys 660 301989DNAArtificial
Sequencecoding sequence of Cry1B variant 30atgccttcaa ataggaaaaa
tgagaatgaa attataaatg ctgtatcgaa tcattccgca 60caaatggatc tatcgctaga
tgctcgtatt gaagatagct tgtgtgtagc cgaggtgaac 120aatattgatc
catttgttag cgcatcaaca gtccaaacag gtattagtat agctggtaga
180atattgggcg tattaggtgt gccgtttgct ggacaactag ctagttttta
tagttttctt 240gttggggaat tatggcctag cggcagagat ccatgggaaa
ttttcctgga acatgtcgaa 300caacttataa gacaacaagt aacagaaaat
actaggaata cggctattgc tcgattagaa 360ggtctaggaa gaggctatag
atcttaccag caggctcttg aaacttggtt agataaccga 420aatgatgcaa
gatcaagaag cattattctt gagcgctatg ttgctttaga acttgacatt
480actactgcta taccgctttt cagcatacga aatcaagagg ttccattatt
aatggtatat 540gctcaagctg caaatttaca cctattatta ttgagagacg
catccctttt tggtagtgaa 600tgggggatgt catctgccga tgttaaccaa
tattaccaag aacaaatcag atatacagag 660gaatattcta accattgcgt
acaatggtat aatacagggc taaataactt aagagggaca 720aatgctgaaa
gttggttgcg gtataatcaa ttccgtagag acctaacgtt aggggtatta
780gatttagtag ccctattccc aagctatgac actcgcactt atccaatcaa
tacgagtgct 840cagttaacaa gagaaattta tacagatcca attgggagaa
caaatgcacc ttcaggattt 900gcaagtacga attggtttaa taataatgca
ccatcgtttt ctgccataga ggctgccatt 960ttcaggcctc cgcatctact
tgattttcca gaacaactta caatttacag tgcatcaagc 1020cgttggagta
gcactcaaca tatgaattat tgggtgggac ataggcttaa cttccgccca
1080ataggaggga cattaaatac ctcaacacaa ggacttacta ataatacttc
aattaatcct 1140gtaacattac agtttacgtc tcgtgacgtt tatagaacag
aatcaaatgc agggacaaat 1200atactattta ctactcctgt gaatggagta
ccttgggcta gatttaattt tataaaccct 1260cagaatattt atgaaagagg
cgccactacc tacagtcaac cgtatcaggg agttgggatt 1320caattatttg
attcagaaac tgaattacca ccagaaacaa cagaacgacc aaattatgaa
1380tcatatagtc atagattatc tcatatagga ctaatcatag gaaacacttt
gagagcacca 1440gtctattctt ggacgcatcg tagtgcaact cttacaaata
caattgatcc agagagaatt 1500aatcaaatac ctttagtgaa aggatttaga
gtttgggggg gcacctctgt cattacagga 1560ccaggattta caggagggga
tatccttcga agaaatacct ttggtgattt tgtatctcta 1620caagtcaata
ttaattcacc aattacccaa agataccgtt taagatttcg ttacgcttcc
1680agtagggatg cacgagttat agtattaaca ggagcggcat ccacaggagt
gggaggccaa 1740gttagtgtaa atatgcctct tcagaaaact atggaaatag
gggagaactt aacatctaga 1800acatttagat ataccgattt tagtaatcct
ttttcattta gagctaatcc agatataatt 1860gggataagtg aacaacctct
atttggtgca ggttctatta gtagcggtga actttatata 1920gataaaattg
aaattattct agcagatgca acatttgaag cagaatctga tttagaaaga
1980gcacaaaag 198931655PRTArtificial SequenceCry1B variant 31Met
Pro Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10
15 Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp
20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn
Ile Asn 35 40 45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe
Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu
Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu
His Val Glu Gln Leu Val Arg Gln His Ile 100 105 110 Thr Glu Asn Ala
Arg Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser
Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140
Arg Asp Asn Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145
150 155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile
Asn Asn 165 170 175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala
Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
Gly Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr
Tyr Glu Arg Gln Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr
Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg
Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg
Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265
270 Ser Tyr Asp Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr
275 280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro
Ser Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro
Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro
His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala
Ser Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val
Gly His Arg Leu Asn Phe Arg Pro Ile His Gly 355 360 365 Thr Leu Asn
Thr Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val
Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390
395 400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro
Trp 405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg
Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr
Gln Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn
Ile Arg Leu Ile Ile Ser Asn Thr Leu 465 470 475 480 Arg Ala Pro Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile
Ala Thr Asn Ile Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515
520 525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Tyr Asn Arg
Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr
Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro
Ile Arg Leu Ser Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser
Ser Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln
Ser Arg Asn Phe Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr
Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu
Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635
640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645
650 655 321968DNAArtificial Sequencecoding sequence of Cry1B
variant 32atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat
cccagccgtc 60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga
ctcactctgc 120atcgccgagg gcaacaacat caacccgctc gtcagcgcct
cgaccgtgca gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc
ggagtcccat tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg
cgagctctgg ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg
tcgagcagct ggtcaggcag cacatcacgg agaacgctcg caacacggct
360ctcgccagac tccaaggcct cggagccagc ttcagagcct accagcagtc
cctcgaggac 420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc
tctacaccca gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca
ctcttcgcca tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca
agctgccaac ctccacctcc tgctcctcag agacgctagc 600ctgttcggca
gcgagttcgg actcacgtcg caggagatcc agcgctacta cgagcgccag
660gcggagaaga cccgggagta cagcgactac tgcgcacgct ggtacaacac
cggcctgaac 720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca
accagttccg cagggacctc 780acactcggag tcctcgacct cgtcgcgctg
ttcccgagct acgacacgcg gatctacccg 840atcaacacga gcgcgcagct
cactcgcgag atctacacgg accccatcgg tcgcacgaac 900gctccatccg
gcttcgcctc caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc
960atcgaagctg caatcttccg cccacctcac ctgctggact tcccagagca
gctcaccatc 1020tacagcgcct ccagccgctg gtccagcacg cagcacatga
actactgggt cggccaccgc 1080ctcaacttca ggcctatcca cggtaccctc
aacacctcga cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct
ccagttcacg agccgggacg tctaccgcac tgagagctac 1200gctggcatca
acatcctgct cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac
1260tggaggaacc ctctcaactc cctgcgcgga tcgctcctct acaccatcgg
ctacaccgga 1320gtcggtaccc agctcttcga cagcgagacc gagctcccac
ctgagaccac cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg
aacatccgcc tcatcatctc caacacgctc 1440agagctcccg tctactcctg
gacgcacagg tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca
cccagatccc ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc
1560tccggaccag gcttcaccgg aggagacctc gtccgcctca acaactccgg
caacaacatc 1620tacaaccggg gctacatcga ggtgccgatc cagttcatct
ccacgagcac tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg
atccgcctct ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt
cccagccacc gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct
acttcgagag ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc
1860gtccgcaact tctccgagaa cgccggagtg atcatcgacc gcttcgagtt
catccccgtg 1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196833655PRTArtificial SequenceCry1B variant 33Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln His Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile His Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr
Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn Gly Ser
Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu Val Arg
Leu Asn Asn Ser Gly Asn Asn Ile Tyr Asn Arg Gly 530 535 540 Tyr Ile
Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545 550 555
560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser Val Asn
565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Thr Ile Val Pro Ala Thr
Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe Gly Tyr
Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Val
Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val Ile Ile
Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr Phe Glu
Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
341968DNAArtificial Sequencecoding sequence of Cry1B variant
34atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag cacatcacgg agaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcca cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg caacacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620tacaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gcaccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196835655PRTArtificial SequenceCry1B variant 35Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Met Ile 100 105 110 Thr Leu Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Gly Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
361968DNAArtificial Sequencecoding sequence of Cry1B variant
36atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag atgatcacgc tcaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcgg cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg cggcacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196837655PRTArtificial SequenceCry1B variant 37Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Met Ile 100 105 110 Thr Met Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Gly Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
381968DNAArtificial Sequencecoding sequence of Cry1B variant
38atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag atgatcacga tgaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcgg cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg cggcacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196839655PRTArtificial SequenceCry1B variant 39Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Met Ile 100 105 110 Thr His Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Gly Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
401968DNAArtificial Sequencecoding sequence of Cry1B variant
40atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag atgatcacgc acaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcgg cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg cggcacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196841655PRTArtificial SequenceCry1B variant 41Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln His Ile 100 105 110 Thr Met Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Gly Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
421968DNAArtificial Sequencecoding sequence of Cry1B variant
42atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag cacatcacga tgaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcgg cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg cggcacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196843655PRTArtificial SequenceCry1B variant 43Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln Met Ile 100 105 110 Thr His Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly
Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg
Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270
Ser Tyr Asp Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275
280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser
Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser
Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His
Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser
Ser Arg Trp Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly
His Arg Leu Asn Phe Arg Pro Ile Asn Gly 355 360 365 Thr Leu Asn Thr
Ser Thr His Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr
Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395
400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp
405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly
Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln
Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu
Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile
Arg Leu Ile Ile Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr
Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala
Thr Asn Ile Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe
Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520
525 Asp Leu Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly
530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg
Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile
Arg Leu Ser Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser
Ile Val Pro Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser
Arg Asn Phe Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser
Ala Thr Gly Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn
Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640
Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650
655 441968DNAArtificial Sequencecoding sequence of Cry1B variant
44atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag atgatcacgc acaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcaa cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg caacacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196845655PRTArtificial SequenceCry1B variant 45Met Pro Ser Asn Arg
Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala
Val Ser Asn His Ser Ala Gln Met Asp Leu Ser Leu Asp 20 25 30 Ala
Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40
45 Pro Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu
Ala Ser 65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser
Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln
Leu Val Arg Gln His Ile 100 105 110 Thr Met Asn Ala Arg Asn Thr Ala
Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr
Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asn Ala
Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu
Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170
175 Gln Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe
Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln
Ala Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp
Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala
Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr
Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp
Thr Arg Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg
Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295
300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp
Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp
Ser Ser Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu
Asn Phe Arg Pro Ile Asn Gly 355 360 365 Thr Leu Asn Thr Ser Thr His
Gly Ala Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe
Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly
Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420
425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp
Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile
Ile Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile
Ile Thr Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn
Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu
Val Arg Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540
Tyr Ile Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545
550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Arg Leu Ser
Val Asn 565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro
Ala Thr Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asn Phe
Gly Tyr Phe Glu Ser Arg 595 600 605 Asn Ala Phe Thr Ser Ala Thr Gly
Asn Val Val Gly Val Arg Asn Phe 610 615 620 Ser Glu Asn Ala Gly Val
Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635 640 Thr Ala Thr
Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 655
461968DNAArtificial Sequencecoding sequence of Cry1B variant
46atgccctcca accgcaagaa cgagaacgag ataatcaacg ccctgtcgat cccagccgtc
60tccaaccact ccgcgcagat ggacctctca ctggacgctc gcatcgagga ctcactctgc
120atcgccgagg gcaacaacat caacccgctc gtcagcgcct cgaccgtgca
gactggcatc 180aacatcgccg gtcgcatact cggcgtcctc ggagtcccat
tcgcaggtca gctggcgagc 240ttctacagct tcatcgtcgg cgagctctgg
ccatcaggtc gcgatccctg ggagatcttc 300atggagcacg tcgagcagct
ggtcaggcag cacatcacga tgaacgctcg caacacggct 360ctcgccagac
tccaaggcct cggagccagc ttcagagcct accagcagtc cctcgaggac
420tggctcgaga accgcgacaa cgcgaggacc cggagcgtcc tctacaccca
gtacatcgcg 480ctggagctcg acttcctgaa cgcgatgcca ctcttcgcca
tcaacaacca gcaggtgccg 540ctcctcatgg tctacgccca agctgccaac
ctccacctcc tgctcctcag agacgctagc 600ctgttcggca gcgagttcgg
actcacgtcg caggagatcc agcgctacta cgagcgccag 660gcggagaaga
cccgggagta cagcgactac tgcgcacgct ggtacaacac cggcctgaac
720aacctgcgcg gcacgaacgc tgagagctgg ctccgctaca accagttccg
cagggacctc 780acactcggag tcctcgacct cgtcgcgctg ttcccgagct
acgacacgcg gatctacccg 840atcaacacga gcgcgcagct cactcgcgag
atctacacgg accccatcgg tcgcacgaac 900gctccatccg gcttcgcctc
caccaactgg ttcaacaaca acgcgccgtc gttcagcgcc 960atcgaagctg
caatcttccg cccacctcac ctgctggact tcccagagca gctcaccatc
1020tacagcgcct ccagccgctg gtccagcacg cagcacatga actactgggt
cggccaccgc 1080ctcaacttca ggcctatcaa cggtaccctc aacacctcga
cccacggcgc cacgaacacg 1140tccatcaacc cggtgacgct ccagttcacg
agccgggacg tctaccgcac tgagagctac 1200gctggcatca acatcctgct
cacgacgcca gtgaacggcg tcccgtgggc acgcttcaac 1260tggaggaacc
ctctcaactc cctgcgcgga tcgctcctct acaccatcgg ctacaccgga
1320gtcggtaccc agctcttcga cagcgagacc gagctcccac ctgagaccac
cgagaggccc 1380aactacgaga gctactccca ccgcctgtcg aacatccgcc
tcatcatcgg caacacgctc 1440agagctcccg tctactcctg gacgcacagg
tcagctgacc ggacgaacac catcgcgacg 1500aacatcatca cccagatccc
ggccgtcaag ggcaacttcc tcttcaacgg ctccgtcatc 1560tccggaccag
gcttcaccgg aggagacctc gtccgcctca acaactccgg caacaacatc
1620cagaaccggg gctacatcga ggtgccgatc cagttcatct ccacgagcac
tcggtaccgc 1680gtcagagtgc gctacgcgag cgtcactccg atccgcctct
ccgtcaactg gggcaactcg 1740aacatcttca gctccatcgt cccagccacc
gcgactagcc tcgacaacct gcagtcccgc 1800aacttcggct acttcgagag
ccgcaacgcc ttcacgagcg cgactggcaa cgtcgtcggc 1860gtccgcaact
tctccgagaa cgccggagtg atcatcgacc gcttcgagtt catccccgtg
1920accgcgacct tcgaggccga gtacgacctt gagagagctc aggaggcc
196847655PRTBacillus thuringiensis 47Met Thr Ser Asn Arg Lys Asn
Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser
Asn His Ser Ala Gln Met Asp Leu Ser Pro Asp 20 25 30 Ala Arg Ile
Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40 45 Pro
Leu Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55
60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser
65 70 75 80 Phe Tyr Ser Phe Ile Val Gly Glu Leu Trp Pro Ser Gly Arg
Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Val
Arg Gln Gln Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala
Arg Leu Gln Gly Leu Gly 115 120 125 Ala Ser Phe Arg Ala Tyr Gln Gln
Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asp Ala Arg Thr
Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu Glu Leu
Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Asn Asn 165 170 175 Gln
Gln Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu
195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Ala Glu
Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser
Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Ile Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile
Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe
Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310
315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro
Glu 325 330 335 Gln Leu Thr Ile Phe Ser Val Leu Ser Arg Trp Ser Asn
Thr Gln Tyr 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Glu Ser
Arg Thr Ile Arg Gly 355 360 365 Ser Leu Ser Thr Ser Thr His Gly Asn
Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr Leu Gln Phe Thr Ser
Arg Asp Val Tyr Arg Thr Glu Ser Tyr 385 390 395 400 Ala Gly Ile Asn
Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp 405 410 415 Ala Arg
Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu 420 425 430
Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser 435
440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu
Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly
Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr Ser Trp Thr His Arg
Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ala Thr Asn Ile Ile Thr
Gln Ile Pro Ala Val Lys Gly Asn 500 505 510 Phe Leu Phe Asn Gly Ser
Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515 520 525 Asp Leu Val Arg
Leu Asn Asn Ser Gly Asn Asn Ile Gln Asn Arg Gly 530 535 540 Tyr Leu
Glu Val Pro Ile Gln Phe Ile Ser Thr Ser Thr Arg Tyr Arg 545 550 555
560 Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile Gln Leu Ser Val Asn
565 570 575 Trp Gly Asn Ser Asn Ile Phe Ser Ser Ile Val Pro Ala Thr
Ala Thr 580 585 590 Ser Leu Asp Asn Leu Gln Ser Arg Asp Phe Gly Tyr
Phe Glu Ser Thr 595
600 605 Asn Ala Phe Thr Ser Ala Thr Gly Asn Val Val Gly Val Arg Asn
Phe 610 615 620 Ser Glu Asn Ala Gly Val Ile Ile Asp Arg Phe Glu Phe
Ile Pro Val 625 630 635 640 Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu
Glu Arg Ala Gln Glu 645 650 655 481965DNABacillus thuringiensis
48atgacttcaa ataggaaaaa tgagaatgaa attataaatg ctttatcgat tccagctgta
60tcgaatcatt ccgcacaaat ggatctatca ccagatgctc gtattgagga ttctttgtgt
120atagccgagg ggaataatat caatccactt gttagcgcat caacagtcca
aacgggtatt 180aacattgctg gtagaatact aggcgtatta ggcgtaccgt
ttgctggaca actagctagt 240ttttatagtt ttattgtcgg tgaattatgg
cctagcggca gagatccgtg ggaaatcttt 300ctagaacatg ttgaacaact
tgtaagacaa caaataacag aaaatgctag gaatacggca 360cttgctcgat
tacaaggttt aggagcttcc tttagagcct atcaacaatc acttgaagac
420tggctagaaa accgtgatga tgcaagaacg agaagtgttc tttataccca
atatatagcc 480ttagagcttg attttcttaa tgcgatgccg cttttcgcaa
taaacaatca acaggttcca 540ttattgatgg tatatgctca agctgcaaat
ttacatctat tattattgag agatgcctct 600ctttttggta gtgaatttgg
gcttacatcg caggaaattc aacgttatta tgagcgccaa 660gcggaaaaaa
cgagagaata ttctgattat tgcgcaagat ggtataatac gggtttaaat
720aatttgagag ggacaaatgc tgaaagttgg ttgcgatata atcaattccg
tagagactta 780acgctaggag tattagatct agtggcacta ttcccaagct
atgacacgcg tatttatcca 840ataaatacca gtgctcaatt aacaagagaa
atttatacag atccaattgg gagaacaaat 900gcaccttcag gatttgcaag
tacgaattgg tttaataata atgcaccatc gttttctgcc 960atagaggctg
ccgttattag gcctccgcat ctacttgatt ttccagaaca gcttacaatt
1020ttcagcgtat taagtcgatg gagtaatact caatatatga attactgggt
gggacataga 1080cttgaatcgc gaacaataag ggggtcatta agtacctcga
cacacggaaa taccaatact 1140tctattaatc ctgtaacatt acagttcaca
tctcgtgacg tttatagaac agaatcatat 1200gcagggataa atatacttct
aactactcct gtgaatggag taccttgggc tagatttaat 1260tggagaaatc
ccctgaattc tcttagaggt agccttctct atactatagg gtatactgga
1320gtggggacac aactatttga ttcagaaact gaattaccac cagaaacaac
agaacgacca 1380aattatgaat cttacagtca tagattatct aatataagac
taatatcagg aaacactttg 1440agagcaccag tatattcttg gacgcaccgt
agtgcagatc gtacgaatac gattgctaca 1500aatattatta ctcaaattcc
tgcagtgaag ggaaactttc tttttaatgg ttctgtaatt 1560tcaggaccag
gatttactgg tggggactta gttagattaa ataatagtgg aaataatatt
1620caaaatagag gctaccttga ggttccgatt caattcatct ccacatctac
cagatatcga 1680gttcgtgtac gttatgcttc tgtaaccccg attcaactca
gtgttaattg gggtaattca 1740aacatttttt ccagcatagt accagctaca
gctacgtcat tagataatct acaatcaagg 1800gattttggtt attttgaaag
taccaatgca tttacatctg caacaggtaa tgtagtaggt 1860gttagaaatt
ttagtgagaa tgcaggagtg ataatagaca gatttgaatt tatcccagtt
1920actgcaacct tcgaagcaga atatgattta gaaagagcgc aagag
196549650PRTBacillus thuringiensis 49Met Pro Ser Asn Arg Lys Asn
Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser
Asn His Ser Ala Gln Met Asp Leu Ser Pro Asp 20 25 30 Ala Arg Ile
Glu Asp Ser Leu Cys Val Ala Glu Gly Asn Asn Ile Asp 35 40 45 Pro
Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Ser Ile Ala Gly 50 55
60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser
65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg
Asp Pro 85 90 95 Trp Glu Ile Phe Met Glu His Val Glu Gln Ile Val
Arg Gln Gln Ile 100 105 110 Thr Asp Ser Val Arg Asp Thr Ala Ile Ala
Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln
Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser
Arg Ser Ile Ile Arg Glu Arg Tyr Ile Ala 145 150 155 160 Leu Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Ser Ile Arg Asn 165 170 175 Gln
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met
195 200 205 Ser Ser Ala Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg
Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Arg Leu Arg Gly Thr Thr Ala Glu Ser
Trp Val Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Thr Tyr Pro Ile Pro Thr Thr Ala Gln Leu Thr 275 280 285 Arg Glu Val
Tyr Thr Asp Pro Asn Gly Val Val Ala Gly Pro Asn Asn 290 295 300 Ser
Trp Phe Arg Asn Gly Ala Ser Phe Ser Ala Ile Glu Asn Ala Ile 305 310
315 320 Ile Arg Gln Pro His Leu Tyr Asp Phe Leu Thr Asn Leu Thr Ile
Tyr 325 330 335 Thr Arg Arg Ser Gln Val Gly Thr Thr Ile Met Asn Leu
Trp Ala Gly 340 345 350 His Arg Ile Thr Phe Asn Arg Ile Gln Gly Gly
Ser Thr Ser Glu Met 355 360 365 Val Tyr Gly Ala Ile Thr Asn Pro Val
Ser Val Ser Asp Ile Pro Phe 370 375 380 Val Asn Arg Asp Val Tyr Arg
Thr Val Ser Leu Ala Gly Gly Leu Gly 385 390 395 400 Ser Leu Ser Gly
Ile Arg Tyr Gly Leu Thr Arg Val Asp Phe Asp Met 405 410 415 Ile Phe
Arg Asn His Pro Asp Ile Val Thr Gly Leu Phe Tyr His Pro 420 425 430
Gly His Ala Gly Ile Ala Thr Gln Val Lys Asp Ser Glu Thr Glu Leu 435
440 445 Pro Pro Glu Thr Thr Glu Gln Pro Asn Tyr Arg Ala Phe Ser His
Leu 450 455 460 Leu Ser His Ile Ser Met Gly Pro Thr Thr Gln Asp Val
Pro Pro Val 465 470 475 480 Tyr Ser Trp Thr His Gln Ser Ala Asp Arg
Thr Asn Thr Ile Asn Ser 485 490 495 Asp Arg Ile Thr Gln Ile Pro Leu
Val Lys Ala His Thr Leu Gln Ser 500 505 510 Gly Thr Thr Val Val Lys
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu 515 520 525 Arg Arg Thr Ser
Gly Gly Pro Phe Ala Phe Ser Asn Val Asn Leu Asp 530 535 540 Phe Asn
Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr 545 550 555
560 Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala
565 570 575 Gly Gln Phe Asp Lys Thr Met Asp Ala Gly Ala Pro Leu Thr
Phe Gln 580 585 590 Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr
Phe Pro Glu Arg 595 600 605 Ser Ser Ser Leu Thr Val Gly Ala Asp Thr
Phe Ser Ser Gly Asn Glu 610 615 620 Val Tyr Val Asp Arg Phe Glu Leu
Ile Pro Val Thr Ala Thr Phe Glu 625 630 635 640 Ala Glu Ser Asp Leu
Glu Arg Ala Arg Lys 645 650 501950DNABacillus thuringiensis
50atgccttcaa ataggaaaaa tgagaatgaa attataaatg ctttatcgat tccagctgta
60tcgaatcatt ccgcacaaat ggatctatca ccagatgctc gcattgagga tagcttgtgt
120gtagccgagg ggaacaatat tgatccattt gttagcgcat caacagtcca
aacaggtatt 180agtatagctg gtagaatatt aggcgtatta ggggtgccgt
ttgccggaca actagctagt 240ttttatagtt ttcttgttgg ggaattatgg
cctagcggca gagatccatg ggaaattttt 300atggaacatg tcgaacaaat
tgtaagacaa caaataacgg acagtgttag ggataccgct 360attgctcgtt
tagaaggtct aggaagaggg tatagatctt accagcaggc tcttgaaact
420tggttagata accgaaatga tgcaagatca agaagcatta ttcgtgagag
atatattgct 480ttagaacttg acattactac tgctataccg cttttcagca
tacgaaatca agaggttcca 540ttattaatgg tatatgctca agctgcaaat
ttacacctat tattattgag agacgcatcc 600ctttttggta gtgaatgggg
gatgtcatct gccgatgtta accaatatta ccaagaacaa 660atcagatata
cagaggaata ttctaaccat tgcgtacaat ggtataatac agggctaaat
720agattaagag ggacaactgc cgaaagttgg gtacggtata atcaattccg
tagagaccta 780acattaggtg tattagattt agtggcacta ttcccaagct
atgacactcg gacttatccc 840attccaacta ccgcccaact tacaagagaa
gtgtatacag atccaaacgg tgttgtagca 900ggacccaata atagttggtt
tagaaatgga gcttcgtttt ccgctataga aaacgcaatt 960attcgacaac
ctcacctata tgattttcta acgaacctta caatttacac gagaagaagt
1020caagtaggca ctacaattat gaatttgtgg gcagggcata gaatcacgtt
taatagaata 1080caaggtggtt ctactagtga aatggtgtat ggggctatta
ctaacccagt tagtgttagt 1140gacataccat ttgtcaatcg ggatgtttac
cgaactgtat cattagctgg tgggcttggc 1200tctctgagtg gaatacgtta
tggtttaact agagttgatt ttgatatgat atttcgtaac 1260catcctgata
tagtaactgg attattttat catccgggac acgcgggcat tgcaacccaa
1320gtaaaagatt cagaaacaga attaccacct gaaacgacag aacagccaaa
ttatagagca 1380tttagtcatc tactaagtca tatttcaatg ggtccaacga
ctcaagacgt acctccagta 1440tattcttgga cacaccagag tgcagatcgt
acgaatacaa tcaattcgga taggataaca 1500caaataccat tggtaaaggc
gcataccctc caatcgggta ccactgtagt aaaagggcca 1560gggtttacag
gaggggatat cctccgtcga acaagtggag gaccatttgc ttttagtaat
1620gttaatctag attttaactt gtcacaaagg tatcgtgcta gaattcgtta
tgcctctact 1680actaacctaa gaatttacgt aacggttgca ggtgaacgaa
tttttgctgg tcaatttgac 1740aaaactatgg atgctggtgc cccattaaca
ttccaatctt ttagttacgc aactattaat 1800acagctttta cattcccaga
aagatcgagc agcttgactg taggtgccga tacgtttagt 1860tcaggtaatg
aagtttatgt agatagattt gaattaatcc cagttactgc aaccttcgag
1920gcagaatctg atttagaaag agcgcggaag 1950513771DNABacillus
thuringiensis 51ttgacttcaa ataggaaaaa tgagaatgaa attataaatg
ctttatcgat tccaacggta 60tcgaatcctt ccacgcaaat gaatctatca ccagatgctc
gtattgaaga tagcttgtgt 120gtagccgagg tgaacaatat tgatccattt
gttagcgcat caacagtcca aacgggtata 180aacatagctg gtagaatatt
gggcgtatta ggtgtgccgt ttgctggaca actagctagt 240ttttatagtt
ttcttgttgg tgaattatgg cctagtggca gagatccatg ggaaattttc
300ctggaacatg tagaacaact tataagacaa caagtaacag aaaatactag
gaatacggct 360attgctcgat tagaaggtct aggaagaggc tatagatctt
accagcaggc tcttgaaact 420tggttagata accgaaatga tgcaagatca
agaagcatta ttcttgagcg ctatgttgct 480ttagaacttg acattactac
tgctataccg cttttcagaa tacgaaatca agaggttcca 540ttattaatgg
tatatgctca agctgcaaat ttacacctat tattattgag ggacgcatcc
600ctttttggta gtgaatgggg gacggcatct tccgatgtta accaatatta
ccaagaacaa 660atcagatata cagaggaata ttctaaccat tgcgtacaat
ggtataatac ggggctaaat 720aacttaagag ggacaaatgc tgaaagttgg
gtacggtata atcaattccg cagagaccta 780acattagggg tattagatct
agtggcccta ttcccaagtt atgacactcg cacttatcca 840atcaatacga
gtgctcagtt aacaagagaa gtttatacag acgcaattgg gaccgtacat
900ccgagtcaag cttttgcaag tacgacttgg tttaataata atgcaccatc
gttttctgcc 960atagaagctg ccgttatcag gcctccgcat ctacttgatt
ttccagaaca acttacaatt 1020tacagcacat taagtcgatg gagtaacact
cagtttatga atatatgggc aggtcataga 1080cttgaatccc gcccaatagc
agggtcatta aatacctcta cacaaggatc taccaatact 1140tctattaatc
ctgtaacatt acagtttacg tctcgagaca tttataggac tgaatcattg
1200gcagggctaa atatatttat aactcaacct gttaatgggg ttccttgggt
tagatttaat 1260tggagaaatc ccctgaattc tcttagaggt agccttctct
atacgatagg gtatactgga 1320gttgggacgc aattacaaga ttcagaaact
gaattacccc cagaaacaac agaacgacca 1380aattatgaat catatagtca
tagattatct catataggac tcatttcatc atctcatgtg 1440agagcattgg
tatattcttg gacgcaccgt agtgcagatc gtacgaatac gattggacca
1500aatagaatta ctcaaattcc tgcagtgaag ggaagatttc tttttaatgg
ctctgtaatt 1560tcaggaccag gatttactgg tggagacgta gttagattga
ataggaataa tggtaatatt 1620caaaatagag ggtatattga agttccaatt
caattcacgt cgacatctac cagatatcga 1680gttcgagtac gttatgcttc
tgtaacctcg attgagctca atgttaattg gggcaattca 1740tcaattttta
cgaacacatt accagcaaca gctgcatcat tagataatct acaatcaggg
1800gattttggtt atgttgaaat caacaatgct tttacatccg caacaggtaa
tatagtaggt 1860gttagaaatt ttagtgcaaa tgcagaggta ataatagaca
gatttgaatt tatcccagtt 1920actgcaacct tcgaggcaaa atatgattta
gaaagagcac aaaaggcggt gaatgctctg 1980tttacttcta caaatccaag
aagattgaag acagatgtga cagattatca tattgaccaa 2040gtgtccaatc
tggtggtatg tttatcagat gaattttgct tggatgagaa gcgagaatta
2100tttgagaaag tgaaatatgc gaagcgactc agtgatgaaa gaaacttact
ccaagatcca 2160aacttcacat tcatcaatgg gcaaccaagt tttgcatcca
tcgatggaca atcaaacttc 2220acctctatta atgagctatc taatcatgga
tggtggggca gtgcgaatgt taccattcag 2280gaagggaatg acgtatttaa
agagaattac gtcacactac cgggtacttt taatgagtgt 2340tatccaaatt
atttatatca aaaaatagga gagtcagaat taaaggctta tacgcgctat
2400caattaagag ggtatattga agatagtcaa gatctagaga tttatttaat
tcgttacaat 2460gcaaagcatg aaacattaaa tgttccaggt accgagtccc
tatggccgct ttcagttgaa 2520agcccaatcg gaaggtgcgg agaaccaaat
cgatgcgcac cacattttgg atggaatcct 2580gatctagatt gttcctgcag
agatagagaa aaatgtgcgc atcattccca tcatttcact 2640ttggatattg
atgttggatg cacagacttg caagaggatc taggcgtgtg ggttgtattc
2700aagattaaga cgcaggaagg ttatgcaaga ttaggaaatc tggaatttat
cgaagagaaa 2760ccattaattg gagaagcact gtctcgtgtg aagagagcgg
aaaaaaaatg gagagacaaa 2820agggaaaaac tacaagtgga aacaaaacga
gtatatatag acgcaaaaga agctgtggat 2880gctttattcg tagattctca
atatgataga ttacaagcag atacaaacat cggtatgatt 2940catgcggcag
atagacttgt tcatcggatc cacgaggctt atcttccaga actacctttc
3000attccaggaa taaatgtggt gatttttgaa gaattagaaa accgtatttc
tactgcattt 3060tccttatatg atgcgagaaa tgtcattaaa aatggcgatt
tcaataatgg attgacatgc 3120tggaacgtga aagggcatgt agaggtacag
cagctgaaca atcatcgttc ggtccttgtc 3180atcccggaat gggaagcaga
agtttcacaa aaggtgcgcg tctgtccagg tcgtggctat 3240attcttcgtg
tcacagcgta caaagaggga tatggggaag gctgcgtaac tattcatgaa
3300gtcgataata atacagacca attgaagttt agcaactgtg agaaaggaca
agtatatcca 3360ggtaatacga tagcatgtaa tgattataat aagaatcatg
gtgcgaatgc atgtagttct 3420cgtaatcgtg gatatgacga attctatgga
aacaccccag ctgattattc tgcaaatcaa 3480aaagaatacg ggggtgcgta
cacttcccac aatcatgcat atggcgaatc ttatgaaagt 3540aattcgtcca
taccagctga ttatgcgccg gtttatgaag aagaagcgta tacacatgga
3600cgaagaggta attcttgtga atataacaga gggtatacac cattaccagc
tggttatgtg 3660acagcagagt tagaatactt cccagaaacg gatacagtat
gggttgagat tggagaaacg 3720gaaggaacat ttatcgtgga caatgtggaa
ttactcctta tggaggaata g 3771521256PRTBacillus thuringiensis 52Met
Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10
15 Ile Pro Thr Val Ser Asn Pro Ser Thr Gln Met Asn Leu Ser Pro Asp
20 25 30 Ala Arg Ile Glu Asp Ser Leu Cys Val Ala Glu Val Asn Asn
Ile Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe
Ala Gly Gln Leu Ala Ser 65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu
Leu Trp Pro Ser Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu
His Val Glu Gln Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr
Arg Asn Thr Ala Ile Ala Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly
Tyr Arg Ser Tyr Gln Gln Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140
Arg Asn Asp Ala Arg Ser Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala 145
150 155 160 Leu Glu Leu Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile
Arg Asn 165 170 175 Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala
Ala Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe
Gly Ser Glu Trp Gly Thr 195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr
Tyr Gln Glu Gln Ile Arg Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His
Cys Val Gln Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg
Gly Thr Asn Ala Glu Ser Trp Val Arg Tyr Asn Gln Phe 245 250 255 Arg
Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265
270 Ser Tyr Asp Thr Arg Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr
275 280 285 Arg Glu Val Tyr Thr Asp Ala Ile Gly Thr Val His Pro Ser
Gln Ala 290 295 300 Phe Ala Ser Thr Thr Trp Phe Asn Asn Asn Ala Pro
Ser Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro
His Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Thr
Leu Ser Arg Trp Ser Asn Thr Gln Phe 340 345 350 Met Asn Ile Trp Ala
Gly His Arg Leu Glu Ser Arg Pro Ile Ala Gly 355 360 365 Ser Leu Asn
Thr Ser Thr Gln Gly Ser Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val
Thr Leu Gln Phe Thr Ser Arg Asp Ile Tyr Arg Thr Glu Ser Leu 385
390
395 400 Ala Gly Leu Asn Ile Phe Ile Thr Gln Pro Val Asn Gly Val Pro
Trp 405 410 415 Val Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg
Gly Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr
Gln Leu Gln Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr
Glu Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser His
Ile Gly Leu Ile Ser Ser Ser His Val 465 470 475 480 Arg Ala Leu Val
Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile
Gly Pro Asn Arg Ile Thr Gln Ile Pro Ala Val Lys Gly Arg 500 505 510
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 515
520 525 Asp Val Val Arg Leu Asn Arg Asn Asn Gly Asn Ile Gln Asn Arg
Gly 530 535 540 Tyr Ile Glu Val Pro Ile Gln Phe Thr Ser Thr Ser Thr
Arg Tyr Arg 545 550 555 560 Val Arg Val Arg Tyr Ala Ser Val Thr Ser
Ile Glu Leu Asn Val Asn 565 570 575 Trp Gly Asn Ser Ser Ile Phe Thr
Asn Thr Leu Pro Ala Thr Ala Ala 580 585 590 Ser Leu Asp Asn Leu Gln
Ser Gly Asp Phe Gly Tyr Val Glu Ile Asn 595 600 605 Asn Ala Phe Thr
Ser Ala Thr Gly Asn Ile Val Gly Val Arg Asn Phe 610 615 620 Ser Ala
Asn Ala Glu Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val 625 630 635
640 Thr Ala Thr Phe Glu Ala Lys Tyr Asp Leu Glu Arg Ala Gln Lys Ala
645 650 655 Val Asn Ala Leu Phe Thr Ser Thr Asn Pro Arg Arg Leu Lys
Thr Asp 660 665 670 Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu
Val Val Cys Leu 675 680 685 Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg
Glu Leu Phe Glu Lys Val 690 695 700 Lys Tyr Ala Lys Arg Leu Ser Asp
Glu Arg Asn Leu Leu Gln Asp Pro 705 710 715 720 Asn Phe Thr Phe Ile
Asn Gly Gln Pro Ser Phe Ala Ser Ile Asp Gly 725 730 735 Gln Ser Asn
Phe Thr Ser Ile Asn Glu Leu Ser Asn His Gly Trp Trp 740 745 750 Gly
Ser Ala Asn Val Thr Ile Gln Glu Gly Asn Asp Val Phe Lys Glu 755 760
765 Asn Tyr Val Thr Leu Pro Gly Thr Phe Asn Glu Cys Tyr Pro Asn Tyr
770 775 780 Leu Tyr Gln Lys Ile Gly Glu Ser Glu Leu Lys Ala Tyr Thr
Arg Tyr 785 790 795 800 Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp
Leu Glu Ile Tyr Leu 805 810 815 Ile Arg Tyr Asn Ala Lys His Glu Thr
Leu Asn Val Pro Gly Thr Glu 820 825 830 Ser Leu Trp Pro Leu Ser Val
Glu Ser Pro Ile Gly Arg Cys Gly Glu 835 840 845 Pro Asn Arg Cys Ala
Pro His Phe Gly Trp Asn Pro Asp Leu Asp Cys 850 855 860 Ser Cys Arg
Asp Arg Glu Lys Cys Ala His His Ser His His Phe Thr 865 870 875 880
Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Gln Glu Asp Leu Gly Val 885
890 895 Trp Val Val Phe Lys Ile Lys Thr Gln Glu Gly Tyr Ala Arg Leu
Gly 900 905 910 Asn Leu Glu Phe Ile Glu Glu Lys Pro Leu Ile Gly Glu
Ala Leu Ser 915 920 925 Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp
Lys Arg Glu Lys Leu 930 935 940 Gln Val Glu Thr Lys Arg Val Tyr Ile
Asp Ala Lys Glu Ala Val Asp 945 950 955 960 Ala Leu Phe Val Asp Ser
Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn 965 970 975 Ile Gly Met Ile
His Ala Ala Asp Arg Leu Val His Arg Ile His Glu 980 985 990 Ala Tyr
Leu Pro Glu Leu Pro Phe Ile Pro Gly Ile Asn Val Val Ile 995 1000
1005 Phe Glu Glu Leu Glu Asn Arg Ile Ser Thr Ala Phe Ser Leu Tyr
1010 1015 1020 Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn
Gly Leu 1025 1030 1035 Thr Cys Trp Asn Val Lys Gly His Val Glu Val
Gln Gln Leu Asn 1040 1045 1050 Asn His Arg Ser Val Leu Val Ile Pro
Glu Trp Glu Ala Glu Val 1055 1060 1065 Ser Gln Lys Val Arg Val Cys
Pro Gly Arg Gly Tyr Ile Leu Arg 1070 1075 1080 Val Thr Ala Tyr Lys
Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile 1085 1090 1095 His Glu Val
Asp Asn Asn Thr Asp Gln Leu Lys Phe Ser Asn Cys 1100 1105 1110 Glu
Lys Gly Gln Val Tyr Pro Gly Asn Thr Ile Ala Cys Asn Asp 1115 1120
1125 Tyr Asn Lys Asn His Gly Ala Asn Ala Cys Ser Ser Arg Asn Arg
1130 1135 1140 Gly Tyr Asp Glu Phe Tyr Gly Asn Thr Pro Ala Asp Tyr
Ser Ala 1145 1150 1155 Asn Gln Lys Glu Tyr Gly Gly Ala Tyr Thr Ser
His Asn His Ala 1160 1165 1170 Tyr Gly Glu Ser Tyr Glu Ser Asn Ser
Ser Ile Pro Ala Asp Tyr 1175 1180 1185 Ala Pro Val Tyr Glu Glu Glu
Ala Tyr Thr His Gly Arg Arg Gly 1190 1195 1200 Asn Ser Cys Glu Tyr
Asn Arg Gly Tyr Thr Pro Leu Pro Ala Gly 1205 1210 1215 Tyr Val Thr
Ala Glu Leu Glu Tyr Phe Pro Glu Thr Asp Thr Val 1220 1225 1230 Trp
Val Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Asn 1235 1240
1245 Val Glu Leu Leu Leu Met Glu Glu 1250 1255 533714DNABacillus
thuringiensis 53ttgaattcaa ataggaaaaa tgagaacgaa attatagatg
cttcatttat tcccgcagta 60tccaatgagt ctgttacaat ctctaaagaa tatgcacaaa
caaatcaatt acaaaacaat 120agcattgagg atggtttgtg tatagccgaa
ggggaatata ttgatccatt tgttagcgca 180tcaacagtcc aaacggggat
tagtatcgct ggtagaatat tgggtgtatt aggtgtgccg 240tttgccggac
aattagctag tttttatagt tttattgttg gtgaattatg gcctaaaggc
300agagaccaat gggaaatttt tatggaacat gtagaacaac ttgtaagaca
acaaataaca 360gcaaatgcta ggaatacggc ccttgctcga ttacaaggtt
taggagattc ctttagagcc 420tatcaacagt cacttgaaga ttggctagag
aaccgtaatg atgcaagaac gagaagtgtt 480ctttatactc aatatatagc
cttagagctt gattttctaa atgcgatgcc gcttttcgca 540ataagagagc
aagaggttcc cttattaatg gtatacgctc aagctgcaaa cttgcaccta
600ttattattga gagacgcctc cctttatggt cgtgaatttg ggcttacctc
ccaagaaatt 660caacgttatt atgaacgcca agtagaaaga acgagggact
attctgacca ttgcgtgcaa 720tggtataata cgggtctaaa taacttaaga
gggacaaatg ctgaaagttg ggtgcggtat 780aatcaattcc gtagagacct
aacattaggg gtattagatc tagtggcact attcccaagc 840tatgacactc
gcacttatcc aataaatacg agtgctcagt taacaaggga agtttataca
900gacgcaattg gagcaacagg ggtaaatatg gcaagtatga attggtataa
taataatgca 960ccttcgtttt ccgctataga gactgcggtt atccgaagcc
cgcatctact tgattttcta 1020gaacaactta aaatttttag cgcttcatca
cgatggagta atactaggca tatgacttat 1080tggcgggggc acacgattca
atctcggcca ataagagggg cattaattac ctcgacacac 1140ggaaatacca
atacttctat taaccctgta acattccagt tcccgtcccg agacgtttat
1200aggactgaat catatgcagg agtgcttcta tggggaattt accttgaacc
tattcatggt 1260gttcctactg ttagatttaa ttttaggaac cctcagaata
cttttgaaag aggtactgct 1320aactatagtc aaccctatga gtcacctggg
cttcaattaa aagattcaga aactgaatta 1380ccaccagaaa caacagaacg
accaaattat gaatcatata gtcatagatt atctcacata 1440gggatcattt
tacaaactag gttgaatgta ccggtatatt cttggacgca tcgtagtgca
1500gatcgtacaa atacaattgg accaaataga attactcaaa ttcctgcagt
gaagggaaac 1560cttcttttta atggttctgt aatttcagga ccaggattta
ctggtgggga cttagttaga 1620ttaaataata gtggaaataa tattcaaaat
agaggctatc ttgaggttcc aattcaattc 1680acatcgacat ctaccagata
tcgagttcgt gtacgttatg cttctgtaac cccgattcac 1740ctcagtgtta
attggggtaa ttcaaacatt ttttccagca cagttccagc tacagctgcg
1800tcattagata atctacaatc aagggatttt ggttattttg aaagtaccaa
tgcatttaca 1860tctgtaacag gtaatgtagt aggtgtaaga aattttagtg
aaaatgccag agtgataata 1920gacagatttg aatttattcc agttactgca
accttcgaag cagaatacga tttagaaagg 1980gcgcaagagg cggtgaatgc
tctgtttact aatacgaatc caagaagatt gaaaacagat 2040gtgacagatt
atcatattga tcaagtatcc aatttagtgg cgtgtttatc ggatgaattc
2100tgcttagatg aaaagagaga attacttgag aaagtgaaat atgcgaaacg
actcagtgat 2160gaaagaaact tactccaaga tccaaacttc acatccatca
ataagcaacc agacttcata 2220tctactaatg agcaatcgaa tttcacatct
atccatgaac aatctgaaca tggatggtgg 2280ggaagtgaga acattacaat
ccaggaagga aatgacgtat ttaaagagaa ttacgtcaca 2340ctaccaggta
cttataatga gtgttatccg acgtatttat atcaaaaaat aggagagtcg
2400gaattaaaag cttatactcg ctaccaatta agaggttata ttgaagatag
tcaagattta 2460gagatatatt tgattcgtta taatgcgaaa catgaaacat
tggatgttcc aggtaccgag 2520tccgtatggc cgctttcagt tgaaagccca
atcagaaggt gcggagaacc gaatcgatgc 2580gcaccacatt ttgaatggaa
tcctgatcta gattgttcct gcagagatgg agaaaaatgt 2640gcgcatcatt
cccatcattt ctctttggat attgatgttg gatgcataga cttgcatgag
2700aacctaggcg tgtgggtggt attcaagatt aagacgcagg aaggtcatgc
aagactaggg 2760aacctggaat ttattgaaga gaaaccatta ttaggagaag
cactgtctcg tgtgaagaga 2820gcagagaaaa aatggagaga caaacgtgaa
aaactacaat tggaaacaaa acgagtatat 2880acagaggcaa aagaagctgt
ggatgcttta tttgtagatt ctcaatatga tagattacaa 2940gcggatacaa
acattggcat gattcatgcg gcagataaac ttgttcatcg aattcgagag
3000gcgtatcttt cagaattatc tgttatccca ggtgtaaatg cggaaatttt
tgaagaatta 3060gaaggtcgca ttatcactgc aatctcccta tacgatgcga
gaaatgtcgt taaaaatggt 3120gattttaata atggattagc atgctggaat
gtaaaagggc atgtagatgt acaacagagc 3180catcaccgtt ctgtccttgt
tatcccagaa tgggaagcag aagtgtcaca agcagttcgc 3240gtctgtccgg
ggcgtggcta tatcctccgt gtcacagcgt acaaagaggg atatggagag
3300ggttgtgtaa cgatccatga aatcgagaac aatacagacg aactaaaatt
taaaaactgt 3360gaagaagagg aagtgtatcc aacggataca ggaacgtgta
atgattatac tgcacaccaa 3420ggtacagcag catgtaattc ccgtaatgct
ggatatgagg atgcatatga agttgatact 3480acagcatctg ttaattacaa
accgacttat gaagaagaaa cgtatacaga tgtacgaaga 3540gataatcatt
gtgaatatga cagagggtat gtgaattatc caccagtacc agctggttat
3600atgacaaaag aattagaata cttcccagaa accgataagg tatggattga
gattggagaa 3660acggaaggga agtttattgt agacagcgtg gaattactcc
ttatggagga atag 3714541237PRTBacillus thuringiensis 54Met Asn Ser
Asn Arg Lys Asn Glu Asn Glu Ile Ile Asp Ala Ser Phe 1 5 10 15 Ile
Pro Ala Val Ser Asn Glu Ser Val Thr Ile Ser Lys Glu Tyr Ala 20 25
30 Gln Thr Asn Gln Leu Gln Asn Asn Ser Ile Glu Asp Gly Leu Cys Ile
35 40 45 Ala Glu Gly Glu Tyr Ile Asp Pro Phe Val Ser Ala Ser Thr
Val Gln 50 55 60 Thr Gly Ile Ser Ile Ala Gly Arg Ile Leu Gly Val
Leu Gly Val Pro 65 70 75 80 Phe Ala Gly Gln Leu Ala Ser Phe Tyr Ser
Phe Ile Val Gly Glu Leu 85 90 95 Trp Pro Lys Gly Arg Asp Gln Trp
Glu Ile Phe Met Glu His Val Glu 100 105 110 Gln Leu Val Arg Gln Gln
Ile Thr Ala Asn Ala Arg Asn Thr Ala Leu 115 120 125 Ala Arg Leu Gln
Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gln Gln Ser 130 135 140 Leu Glu
Asp Trp Leu Glu Asn Arg Asn Asp Ala Arg Thr Arg Ser Val 145 150 155
160 Leu Tyr Thr Gln Tyr Ile Ala Leu Glu Leu Asp Phe Leu Asn Ala Met
165 170 175 Pro Leu Phe Ala Ile Arg Glu Gln Glu Val Pro Leu Leu Met
Val Tyr 180 185 190 Ala Gln Ala Ala Asn Leu His Leu Leu Leu Leu Arg
Asp Ala Ser Leu 195 200 205 Tyr Gly Arg Glu Phe Gly Leu Thr Ser Gln
Glu Ile Gln Arg Tyr Tyr 210 215 220 Glu Arg Gln Val Glu Arg Thr Arg
Asp Tyr Ser Asp His Cys Val Gln 225 230 235 240 Trp Tyr Asn Thr Gly
Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser 245 250 255 Trp Val Arg
Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu Gly Val Leu 260 265 270 Asp
Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro Ile 275 280
285 Asn Thr Ser Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala Ile Gly
290 295 300 Ala Thr Gly Val Asn Met Ala Ser Met Asn Trp Tyr Asn Asn
Asn Ala 305 310 315 320 Pro Ser Phe Ser Ala Ile Glu Thr Ala Val Ile
Arg Ser Pro His Leu 325 330 335 Leu Asp Phe Leu Glu Gln Leu Lys Ile
Phe Ser Ala Ser Ser Arg Trp 340 345 350 Ser Asn Thr Arg His Met Thr
Tyr Trp Arg Gly His Thr Ile Gln Ser 355 360 365 Arg Pro Ile Arg Gly
Ala Leu Ile Thr Ser Thr His Gly Asn Thr Asn 370 375 380 Thr Ser Ile
Asn Pro Val Thr Phe Gln Phe Pro Ser Arg Asp Val Tyr 385 390 395 400
Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu Trp Gly Ile Tyr Leu Glu 405
410 415 Pro Ile His Gly Val Pro Thr Val Arg Phe Asn Phe Arg Asn Pro
Gln 420 425 430 Asn Thr Phe Glu Arg Gly Thr Ala Asn Tyr Ser Gln Pro
Tyr Glu Ser 435 440 445 Pro Gly Leu Gln Leu Lys Asp Ser Glu Thr Glu
Leu Pro Pro Glu Thr 450 455 460 Thr Glu Arg Pro Asn Tyr Glu Ser Tyr
Ser His Arg Leu Ser His Ile 465 470 475 480 Gly Ile Ile Leu Gln Thr
Arg Leu Asn Val Pro Val Tyr Ser Trp Thr 485 490 495 His Arg Ser Ala
Asp Arg Thr Asn Thr Ile Gly Pro Asn Arg Ile Thr 500 505 510 Gln Ile
Pro Ala Val Lys Gly Asn Leu Leu Phe Asn Gly Ser Val Ile 515 520 525
Ser Gly Pro Gly Phe Thr Gly Gly Asp Leu Val Arg Leu Asn Asn Ser 530
535 540 Gly Asn Asn Ile Gln Asn Arg Gly Tyr Leu Glu Val Pro Ile Gln
Phe 545 550 555 560 Thr Ser Thr Ser Thr Arg Tyr Arg Val Arg Val Arg
Tyr Ala Ser Val 565 570 575 Thr Pro Ile His Leu Ser Val Asn Trp Gly
Asn Ser Asn Ile Phe Ser 580 585 590 Ser Thr Val Pro Ala Thr Ala Ala
Ser Leu Asp Asn Leu Gln Ser Arg 595 600 605 Asp Phe Gly Tyr Phe Glu
Ser Thr Asn Ala Phe Thr Ser Val Thr Gly 610 615 620 Asn Val Val Gly
Val Arg Asn Phe Ser Glu Asn Ala Arg Val Ile Ile 625 630 635 640 Asp
Arg Phe Glu Phe Ile Pro Val Thr Ala Thr Phe Glu Ala Glu Tyr 645 650
655 Asp Leu Glu Arg Ala Gln Glu Ala Val Asn Ala Leu Phe Thr Asn Thr
660 665 670 Asn Pro Arg Arg Leu Lys Thr Asp Val Thr Asp Tyr His Ile
Asp Gln 675 680 685 Val Ser Asn Leu Val Ala Cys Leu Ser Asp Glu Phe
Cys Leu Asp Glu 690 695 700 Lys Arg Glu Leu Leu Glu Lys Val Lys Tyr
Ala Lys Arg Leu Ser Asp 705 710 715 720 Glu Arg Asn Leu Leu Gln Asp
Pro Asn Phe Thr Ser Ile Asn Lys Gln 725 730 735 Pro Asp Phe Ile Ser
Thr Asn Glu Gln Ser Asn Phe Thr Ser Ile His 740 745 750 Glu Gln Ser
Glu His Gly Trp Trp Gly Ser Glu Asn Ile Thr Ile Gln 755 760 765 Glu
Gly Asn Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr 770 775
780 Tyr Asn Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Gly Glu Ser
785 790 795 800 Glu Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr
Ile Glu Asp 805 810 815 Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr
Asn Ala Lys His Glu 820 825 830 Thr Leu Asp Val Pro Gly Thr Glu Ser
Val Trp Pro Leu Ser Val Glu 835 840 845 Ser Pro Ile Arg Arg Cys Gly
Glu Pro Asn Arg Cys Ala Pro
His Phe 850 855 860 Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp
Gly Glu Lys Cys 865 870 875 880 Ala His His Ser His His Phe Ser Leu
Asp Ile Asp Val Gly Cys Ile 885 890 895 Asp Leu His Glu Asn Leu Gly
Val Trp Val Val Phe Lys Ile Lys Thr 900 905 910 Gln Glu Gly His Ala
Arg Leu Gly Asn Leu Glu Phe Ile Glu Glu Lys 915 920 925 Pro Leu Leu
Gly Glu Ala Leu Ser Arg Val Lys Arg Ala Glu Lys Lys 930 935 940 Trp
Arg Asp Lys Arg Glu Lys Leu Gln Leu Glu Thr Lys Arg Val Tyr 945 950
955 960 Thr Glu Ala Lys Glu Ala Val Asp Ala Leu Phe Val Asp Ser Gln
Tyr 965 970 975 Asp Arg Leu Gln Ala Asp Thr Asn Ile Gly Met Ile His
Ala Ala Asp 980 985 990 Lys Leu Val His Arg Ile Arg Glu Ala Tyr Leu
Ser Glu Leu Ser Val 995 1000 1005 Ile Pro Gly Val Asn Ala Glu Ile
Phe Glu Glu Leu Glu Gly Arg 1010 1015 1020 Ile Ile Thr Ala Ile Ser
Leu Tyr Asp Ala Arg Asn Val Val Lys 1025 1030 1035 Asn Gly Asp Phe
Asn Asn Gly Leu Ala Cys Trp Asn Val Lys Gly 1040 1045 1050 His Val
Asp Val Gln Gln Ser His His Arg Ser Val Leu Val Ile 1055 1060 1065
Pro Glu Trp Glu Ala Glu Val Ser Gln Ala Val Arg Val Cys Pro 1070
1075 1080 Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly
Tyr 1085 1090 1095 Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn
Asn Thr Asp 1100 1105 1110 Glu Leu Lys Phe Lys Asn Cys Glu Glu Glu
Glu Val Tyr Pro Thr 1115 1120 1125 Asp Thr Gly Thr Cys Asn Asp Tyr
Thr Ala His Gln Gly Thr Ala 1130 1135 1140 Ala Cys Asn Ser Arg Asn
Ala Gly Tyr Glu Asp Ala Tyr Glu Val 1145 1150 1155 Asp Thr Thr Ala
Ser Val Asn Tyr Lys Pro Thr Tyr Glu Glu Glu 1160 1165 1170 Thr Tyr
Thr Asp Val Arg Arg Asp Asn His Cys Glu Tyr Asp Arg 1175 1180 1185
Gly Tyr Val Asn Tyr Pro Pro Val Pro Ala Gly Tyr Met Thr Lys 1190
1195 1200 Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
Ile 1205 1210 1215 Gly Glu Thr Glu Gly Lys Phe Ile Val Asp Ser Val
Glu Leu Leu 1220 1225 1230 Leu Met Glu Glu 1235 55650PRTBacillus
thuringiensis 55Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn
Ala Val Ser 1 5 10 15 Asn His Ser Ala Gln Met Asp Leu Leu Pro Asp
Ala Arg Ile Glu Asp 20 25 30 Ser Leu Cys Ile Ala Glu Gly Asn Asn
Ile Asp Pro Phe Val Ser Ala 35 40 45 Ser Thr Val Gln Thr Gly Ile
Asn Ile Ala Gly Arg Ile Leu Gly Val 50 55 60 Leu Gly Val Pro Phe
Ala Gly Gln Leu Ala Ser Phe Tyr Ser Phe Leu 65 70 75 80 Val Gly Glu
Leu Trp Pro Arg Gly Arg Asp Gln Trp Glu Ile Phe Leu 85 90 95 Glu
His Val Glu Gln Leu Ile Asn Gln Gln Ile Thr Glu Asn Ala Arg 100 105
110 Asn Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Ala
115 120 125 Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asp Asp
Ala Arg 130 135 140 Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala Leu
Glu Leu Asp Phe 145 150 155 160 Leu Asn Ala Met Pro Leu Phe Ala Ile
Arg Asn Gln Glu Val Pro Leu 165 170 175 Leu Met Val Tyr Ala Gln Ala
Ala Asn Leu His Leu Leu Leu Leu Arg 180 185 190 Asp Ala Ser Leu Phe
Gly Ser Glu Phe Gly Leu Thr Ser Gln Glu Ile 195 200 205 Gln Arg Tyr
Tyr Glu Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp 210 215 220 Tyr
Cys Val Glu Trp Tyr Asn Thr Gly Leu Asn Ser Leu Arg Gly Thr 225 230
235 240 Asn Ala Ala Ser Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu
Thr 245 250 255 Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro Ser Tyr
Asp Thr Arg 260 265 270 Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr
Arg Glu Val Tyr Thr 275 280 285 Asp Ala Ile Gly Ala Thr Gly Val Asn
Met Ala Ser Met Asn Trp Tyr 290 295 300 Asn Asn Asn Ala Pro Ser Phe
Ser Ala Ile Glu Ala Ala Ala Ile Arg 305 310 315 320 Ser Pro His Leu
Leu Asp Phe Leu Glu Gln Leu Thr Ile Phe Ser Ala 325 330 335 Ser Ser
Arg Trp Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His 340 345 350
Thr Ile Gln Ser Arg Pro Ile Gly Gly Gly Leu Asn Thr Ser Thr His 355
360 365 Gly Ala Thr Asn Thr Ser Ile Asn Pro Val Thr Leu Arg Phe Ala
Ser 370 375 380 Arg Asp Val Tyr Arg Thr Glu Ser Tyr Ala Gly Val Leu
Leu Trp Gly 385 390 395 400 Ile Tyr Leu Glu Pro Ile His Gly Val Pro
Thr Val Arg Phe Asn Phe 405 410 415 Thr Asn Pro Gln Asn Ile Ser Asp
Arg Gly Thr Ala Asn Tyr Ser Gln 420 425 430 Pro Tyr Glu Ser Pro Gly
Leu Gln Leu Lys Asp Ser Glu Thr Glu Leu 435 440 445 Pro Pro Glu Thr
Thr Glu Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg 450 455 460 Leu Ser
His Ile Gly Ile Ile Leu Gln Ser Arg Val Asn Val Pro Val 465 470 475
480 Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn Thr Ile Gly Pro
485 490 495 Asn Arg Ile Thr Gln Ile Pro Met Val Lys Ala Ser Glu Leu
Pro Gln 500 505 510 Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly
Gly Asp Ile Leu 515 520 525 Arg Arg Thr Asn Thr Gly Gly Phe Gly Pro
Ile Arg Val Thr Val Asn 530 535 540 Gly Pro Leu Thr Gln Arg Tyr Arg
Ile Gly Phe Arg Tyr Ala Ser Thr 545 550 555 560 Val Asp Phe Asp Phe
Phe Val Ser Arg Gly Gly Thr Thr Val Asn Asn 565 570 575 Phe Arg Phe
Leu Arg Thr Met Asn Ser Gly Asp Glu Leu Lys Tyr Gly 580 585 590 Asn
Phe Val Arg Arg Ala Phe Thr Thr Pro Phe Thr Phe Thr Gln Ile 595 600
605 Gln Asp Ile Ile Arg Thr Ser Ile Gln Gly Leu Ser Gly Asn Gly Glu
610 615 620 Val Tyr Ile Asp Lys Ile Glu Ile Ile Pro Val Thr Ala Thr
Phe Glu 625 630 635 640 Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645
650 56655PRTBacillus thuringiensis 56Met Thr Ser Asn Arg Lys Asn
Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Thr Val Ser
Asn Pro Ser Thr Gln Met Asn Leu Ser Pro Asp 20 25 30 Ala Arg Ile
Glu Asp Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp 35 40 45 Pro
Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55
60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser
65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg
Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile
Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala
Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln
Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser
Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala 145 150 155 160 Leu Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile Arg Asn 165 170 175 Glu
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met
195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg
Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser
Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile
Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe
Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310
315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro
Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser
Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Asn Phe
Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr Gln Gly Leu
Thr Asn Asn Thr Ser Ile Asn 370 375 380 Pro Val Thr Leu Gln Phe Thr
Ser Arg Asp Val Tyr Arg Thr Glu Ser 385 390 395 400 Asn Ala Gly Thr
Asn Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro 405 410 415 Trp Ala
Arg Phe Asn Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly 420 425 430
Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe 435
440 445 Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr 450 455 460 Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile
Ile Gly Asn 465 470 475 480 Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg 485 490 495 Thr Asn Thr Ile Gly Pro Asn Arg
Ile Thr Gln Ile Pro Leu Val Lys 500 505 510 Ala Leu Asn Leu His Ser
Gly Val Thr Val Val Gly Gly Pro Gly Phe 515 520 525 Thr Gly Gly Asp
Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe Gly Asp 530 535 540 Ile Arg
Leu Asn Ile Asn Val Pro Leu Ser Gln Arg Tyr Arg Val Arg 545 550 555
560 Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg Ile Asn
565 570 575 Gly Thr Thr Val Asn Ile Gly Asn Phe Ser Arg Thr Met Asn
Arg Gly 580 585 590 Asp Asn Leu Glu Tyr Arg Ser Phe Arg Thr Ala Gly
Phe Ser Thr Pro 595 600 605 Phe Asn Phe Leu Asn Ala Gln Ser Thr Phe
Thr Leu Gly Ala Gln Ser 610 615 620 Phe Ser Asn Gln Glu Val Tyr Ile
Asp Arg Val Glu Phe Val Pro Ala 625 630 635 640 Glu Val Thr Phe Glu
Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys 645 650 655
57655PRTBacillus thuringiensis 57Met Thr Ser Asn Arg Lys Asn Glu
Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Thr Val Ser Asn
Pro Ser Thr Gln Met Asn Leu Ser Pro Asp 20 25 30 Ala Arg Ile Glu
Asp Ser Leu Cys Val Ala Glu Val Asn Asn Ile Asp 35 40 45 Pro Phe
Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60
Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Leu Ala Ser 65
70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Ser Gly Arg
Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile
Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg Asn Thr Ala Ile Ala
Arg Leu Glu Gly Leu Gly 115 120 125 Arg Gly Tyr Arg Ser Tyr Gln Gln
Ala Leu Glu Thr Trp Leu Asp Asn 130 135 140 Arg Asn Asp Ala Arg Ser
Arg Ser Ile Ile Leu Glu Arg Tyr Val Ala 145 150 155 160 Leu Glu Leu
Asp Ile Thr Thr Ala Ile Pro Leu Phe Arg Ile Arg Asn 165 170 175 Glu
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Trp Gly Met
195 200 205 Ala Ser Ser Asp Val Asn Gln Tyr Tyr Gln Glu Gln Ile Arg
Tyr Thr 210 215 220 Glu Glu Tyr Ser Asn His Cys Val Gln Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly Thr Asn Ala Glu Ser
Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Ile
Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly 290 295 300 Phe
Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala 305 310
315 320 Ile Glu Ala Ala Ile Phe Arg Pro Pro His Leu Leu Asp Phe Pro
Glu 325 330 335 Gln Leu Thr Ile Tyr Ser Ala Ser Ser Arg Trp Ser Ser
Thr Gln His 340 345 350 Met Asn Tyr Trp Val Gly His Arg Leu Asn Phe
Arg Pro Ile Gly Gly 355 360 365 Thr Leu Asn Thr Ser Thr Gln Gly Leu
Thr Asn Asn Thr Ser Ile Asn 370 375 380 Pro Val Thr Leu Gln Phe Thr
Ser Arg Asp Val Tyr Arg Thr Glu Ser 385 390 395 400 Asn Ala Gly Thr
Asn Ile Leu Phe Thr Thr Pro Val Asn Gly Val Pro 405 410 415 Trp Ala
Arg Phe Asn Phe Ile Asn Pro Gln Asn Ile Tyr Glu Arg Gly 420 425 430
Ala Thr Thr Tyr Ser Gln Pro Tyr Gln Gly Val Gly Ile Gln Leu Phe 435
440 445 Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn
Tyr 450 455 460 Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile
Ile Gly Asn 465 470 475 480 Thr Leu Arg Ala Pro Val Tyr Ser Trp Thr
His Arg Ser Ala Asp Arg 485 490 495 Thr Asn Thr Ile Gly Pro Asn Arg
Ile Thr Gln Ile Pro Leu Val Lys 500 505 510 Ala Leu Asn Leu His Ser
Gly Val Thr Val Val Gly Gly Pro Gly Phe 515 520 525 Thr Gly Gly Asp
Ile Leu Arg Arg Thr Asn Thr Gly Thr Phe Gly Asp 530 535 540 Ile Arg
Leu Asn Ile Asn Val Pro Leu Ser Gln Arg Tyr Arg Val Arg 545 550 555
560 Ile Arg Tyr Ala Ser Thr Thr Asp Leu Gln Phe Phe Thr Arg Ile Asn
565 570 575 Gly Thr Thr Val Asn Ile Gly Asn Phe Ser Arg Thr Met Asn
Arg Gly 580 585 590 Asp Asn
Leu Glu Tyr Arg Ser Phe Arg Thr Ala Gly Phe Ser Thr Pro 595 600 605
Phe Asn Phe Leu Asn Ala Gln Ser Thr Phe Thr Leu Gly Ala Gln Ser 610
615 620 Phe Ser Asn Gln Glu Val Tyr Ile Asp Arg Val Glu Phe Val Pro
Ala 625 630 635 640 Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg
Ala Gln Lys 645 650 655 58653PRTBacillus thuringiensis 58Met Thr
Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15
Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asn Leu Ser Thr Asp 20
25 30 Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile
Asp 35 40 45 Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn
Ile Ala Gly 50 55 60 Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala
Gly Gln Ile Ala Ser 65 70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu
Trp Pro Arg Gly Arg Asp Pro 85 90 95 Trp Glu Ile Phe Leu Glu His
Val Glu Gln Leu Ile Arg Gln Gln Val 100 105 110 Thr Glu Asn Thr Arg
Asp Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly 115 120 125 Asn Ser Phe
Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg
Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150
155 160 Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg
Asn 165 170 175 Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala
Asn Leu His 180 185 190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly
Ser Glu Phe Gly Leu 195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr
Glu Arg Gln Val Glu Lys Thr 210 215 220 Arg Glu Tyr Ser Asp Tyr Cys
Ala Arg Trp Tyr Asn Thr Gly Leu Asn 225 230 235 240 Asn Leu Arg Gly
Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe 245 250 255 Arg Arg
Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270
Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gln Leu Thr 275
280 285 Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser
Gly 290 295 300 Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser
Phe Ser Ala 305 310 315 320 Ile Glu Ala Ala Val Ile Arg Pro Pro His
Leu Leu Asp Phe Pro Glu 325 330 335 Gln Leu Thr Ile Phe Ser Val Leu
Ser Arg Trp Ser Asn Thr Gln Tyr 340 345 350 Met Asn Tyr Trp Val Gly
His Arg Leu Glu Ser Arg Thr Ile Arg Gly 355 360 365 Ser Leu Ser Thr
Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro 370 375 380 Val Thr
Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe 385 390 395
400 Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp
405 410 415 Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly
Ser Leu 420 425 430 Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln
Leu Phe Asp Ser 435 440 445 Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu
Arg Pro Asn Tyr Glu Ser 450 455 460 Tyr Ser His Arg Leu Ser Asn Ile
Arg Leu Ile Ser Gly Asn Thr Leu 465 470 475 480 Arg Ala Pro Val Tyr
Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn 485 490 495 Thr Ile Ser
Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ser Phe 500 505 510 Asn
Leu Asn Ser Gly Thr Ser Val Val Ser Gly Pro Gly Phe Thr Gly 515 520
525 Gly Asp Ile Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met Gly
530 535 540 Leu Asn Phe Asn Asn Thr Ser Leu Gln Arg Tyr Arg Val Arg
Val Arg 545 550 555 560 Tyr Ala Ala Ser Gln Thr Met Val Leu Arg Val
Thr Val Gly Gly Ser 565 570 575 Thr Thr Phe Asp Gln Gly Phe Pro Ser
Thr Met Ser Ala Asn Glu Ser 580 585 590 Leu Thr Ser Gln Ser Phe Arg
Phe Ala Glu Phe Pro Val Gly Ile Ser 595 600 605 Ala Ser Gly Ser Gln
Thr Ala Gly Ile Ser Ile Ser Asn Asn Ala Gly 610 615 620 Arg Gln Thr
Phe His Phe Asp Lys Ile Glu Phe Ile Pro Ile Thr Ala 625 630 635 640
Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650
59654PRTBacillus thuringiensis 59Met Thr Ser Asn Arg Lys Asn Glu
Asn Glu Ile Ile Asn Ala Leu Ser 1 5 10 15 Ile Pro Ala Val Ser Asn
His Ser Thr Gln Met Asp Leu Ser Pro Asp 20 25 30 Ala Arg Ile Glu
Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asn 35 40 45 Pro Leu
Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly 50 55 60
Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser 65
70 75 80 Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Arg Gly Arg
Asp Gln 85 90 95 Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile
Asn Gln Gln Ile 100 105 110 Thr Glu Asn Ala Arg Asn Thr Ala Leu Ala
Arg Leu Gln Gly Leu Gly 115 120 125 Asp Ser Phe Arg Ala Tyr Gln Gln
Ser Leu Glu Asp Trp Leu Glu Asn 130 135 140 Arg Asp Asp Ala Arg Thr
Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala 145 150 155 160 Leu Glu Leu
Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn 165 170 175 Gln
Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His 180 185
190 Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu
195 200 205 Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu
Gln Thr 210 215 220 Arg Asp Tyr Ser Asp Tyr Cys Val Glu Trp Tyr Asn
Thr Gly Leu Asn 225 230 235 240 Ser Leu Arg Gly Thr Asn Ala Ala Ser
Trp Val Arg Tyr Asn Gln Phe 245 250 255 Arg Arg Asp Leu Thr Leu Gly
Val Leu Asp Leu Val Ala Leu Phe Pro 260 265 270 Ser Tyr Asp Thr Arg
Thr Tyr Pro Ile Asn Thr Ser Ala Gln Leu Thr 275 280 285 Arg Glu Val
Tyr Thr Asp Ala Ile Gly Ala Thr Gly Val Asn Met Ala 290 295 300 Ser
Met Asn Trp Tyr Asn Asn Asn Ala Pro Ser Phe Ser Ala Ile Glu 305 310
315 320 Thr Ala Val Ile Arg Ser Pro His Leu Leu Asp Phe Leu Glu Gln
Leu 325 330 335 Thr Ile Phe Ser Thr Ser Ser Arg Trp Ser Ala Thr Arg
His Met Thr 340 345 350 Tyr Trp Arg Gly His Thr Ile Gln Ser Arg Pro
Ile Gly Gly Gly Leu 355 360 365 Asn Thr Ser Thr His Gly Ser Thr Asn
Thr Ser Ile Asn Pro Val Arg 370 375 380 Leu Ser Phe Phe Ser Arg Asp
Val Tyr Trp Thr Glu Ser Tyr Ala Gly 385 390 395 400 Val Leu Leu Trp
Gly Ile Tyr Leu Glu Pro Ile His Gly Val Pro Thr 405 410 415 Val Arg
Phe Asn Phe Arg Asn Pro Gln Asn Thr Phe Glu Arg Gly Thr 420 425 430
Ala Asn Tyr Ser Gln Pro Tyr Glu Ser Pro Gly Leu Gln Leu Lys Asp 435
440 445 Ser Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr
Glu 450 455 460 Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile Ser
Gln Ser Arg 465 470 475 480 Val His Val Pro Val Tyr Ser Trp Thr His
Arg Ser Ala Asp Arg Thr 485 490 495 Asn Thr Ile Ser Ser Asp Ser Ile
Thr Gln Ile Pro Leu Val Lys Ser 500 505 510 Phe Asn Leu Asn Ser Gly
Thr Ser Val Val Ser Gly Pro Gly Phe Thr 515 520 525 Gly Gly Asp Ile
Ile Arg Thr Asn Val Asn Gly Ser Val Leu Ser Met 530 535 540 Gly Leu
Asn Phe Asn Asn Thr Ser Leu Gln Arg Tyr Arg Val Arg Val 545 550 555
560 Arg Tyr Ala Ala Ser Gln Thr Met Val Leu Arg Val Thr Val Gly Gly
565 570 575 Ser Thr Thr Phe Asp Gln Gly Phe Pro Ser Thr Met Ser Ala
Asn Glu 580 585 590 Ser Leu Thr Ser Gln Ser Phe Arg Phe Ala Glu Phe
Pro Val Gly Ile 595 600 605 Ser Ala Ser Gly Ser Gln Thr Ala Gly Ile
Ser Ile Ser Asn Asn Ala 610 615 620 Gly Arg Gln Thr Phe His Phe Asp
Lys Ile Glu Phe Ile Pro Ile Thr 625 630 635 640 Ala Thr Phe Glu Ala
Glu Tyr Asp Leu Glu Arg Ala Gln Glu 645 650 60661PRTBacillus
thuringiensis 60Met Asn Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asp
Ala Ser Phe 1 5 10 15 Ile Pro Ala Val Ser Asn Glu Ser Val Thr Ile
Ser Lys Glu Tyr Ala 20 25 30 Gln Thr Asn Gln Leu Gln Asn Asn Ser
Ile Glu Asp Gly Leu Cys Ile 35 40 45 Ala Glu Gly Glu Tyr Ile Asp
Pro Phe Val Ser Ala Ser Thr Val Gln 50 55 60 Thr Gly Ile Ser Ile
Ala Gly Arg Ile Leu Gly Val Leu Gly Val Pro 65 70 75 80 Phe Ala Gly
Gln Leu Ala Ser Phe Tyr Ser Phe Ile Val Gly Glu Leu 85 90 95 Trp
Pro Lys Gly Arg Asp Gln Trp Glu Ile Phe Met Glu His Val Glu 100 105
110 Gln Leu Val Arg Gln Gln Ile Thr Ala Asn Ala Arg Asn Thr Ala Leu
115 120 125 Ala Arg Leu Gln Gly Leu Gly Asp Ser Phe Arg Ala Tyr Gln
Gln Ser 130 135 140 Leu Glu Asp Trp Leu Glu Asn Arg Asn Asp Ala Arg
Thr Arg Ser Val 145 150 155 160 Leu Tyr Thr Gln Tyr Ile Ala Leu Glu
Leu Asp Phe Leu Asn Ala Met 165 170 175 Pro Leu Phe Ala Ile Arg Glu
Gln Glu Val Pro Leu Leu Met Val Tyr 180 185 190 Ala Gln Ala Ala Asn
Leu His Leu Leu Leu Leu Arg Asp Ala Ser Leu 195 200 205 Tyr Gly Arg
Glu Phe Gly Leu Thr Ser Gln Glu Ile Gln Arg Tyr Tyr 210 215 220 Glu
Arg Gln Val Glu Arg Thr Arg Asp Tyr Ser Asp His Cys Val Gln 225 230
235 240 Trp Tyr Asn Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu
Ser 245 250 255 Trp Val Arg Tyr Asn Gln Phe Arg Arg Asp Leu Thr Leu
Gly Val Leu 260 265 270 Asp Leu Val Ala Leu Phe Pro Ser Tyr Asp Thr
Arg Thr Tyr Pro Ile 275 280 285 Asn Thr Ser Ala Gln Leu Thr Arg Glu
Val Tyr Thr Asp Ala Ile Gly 290 295 300 Ala Thr Gly Val Asn Met Ala
Ser Met Asn Trp Tyr Asn Asn Asn Ala 305 310 315 320 Pro Ser Phe Ser
Ala Ile Glu Thr Ala Val Ile Arg Ser Pro His Leu 325 330 335 Leu Asp
Phe Leu Glu Gln Leu Lys Ile Phe Ser Ala Ser Ser Arg Trp 340 345 350
Ser Asn Thr Arg His Met Thr Tyr Trp Arg Gly His Thr Ile Gln Ser 355
360 365 Arg Pro Ile Arg Gly Ala Leu Ile Thr Ser Thr His Gly Asn Thr
Asn 370 375 380 Thr Ser Ile Asn Pro Val Thr Phe Gln Phe Pro Ser Arg
Asp Val Tyr 385 390 395 400 Arg Thr Glu Ser Tyr Ala Gly Val Leu Leu
Trp Gly Ile Tyr Leu Glu 405 410 415 Pro Ile His Gly Val Pro Thr Val
Arg Phe Asn Phe Arg Asn Pro Gln 420 425 430 Asn Thr Phe Glu Arg Gly
Thr Ala Asn Tyr Ser Gln Pro Tyr Glu Ser 435 440 445 Pro Gly Leu Gln
Leu Lys Asp Ser Glu Thr Glu Leu Pro Pro Glu Thr 450 455 460 Thr Glu
Arg Pro Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile 465 470 475
480 Gly Ile Ile Leu Gln Thr Arg Leu Asn Val Pro Val Tyr Ser Trp Thr
485 490 495 His Arg Ser Ala Asp Arg Thr Asn Thr Ile Ser Ser Asp Ser
Ile Thr 500 505 510 Gln Ile Pro Leu Val Lys Ser Phe Asn Leu Asn Ser
Gly Thr Ser Val 515 520 525 Val Ser Gly Pro Gly Phe Thr Gly Gly Asp
Ile Ile Arg Thr Asn Val 530 535 540 Asn Gly Ser Val Leu Ser Met Gly
Leu Asn Phe Asn Asn Thr Ser Leu 545 550 555 560 Gln Arg Tyr Arg Val
Arg Val Arg Tyr Ala Ala Ser Gln Thr Met Val 565 570 575 Leu Arg Val
Thr Val Gly Gly Ser Thr Thr Phe Asp Gln Gly Phe Pro 580 585 590 Ser
Thr Met Ser Ala Asn Glu Ser Leu Thr Ser Gln Ser Phe Arg Phe 595 600
605 Ala Glu Phe Pro Val Gly Ile Ser Ala Ser Gly Ser Gln Thr Ala Gly
610 615 620 Ile Ser Ile Ser Asn Asn Ala Gly Arg Gln Thr Phe His Phe
Asp Lys 625 630 635 640 Ile Glu Phe Ile Pro Ile Thr Ala Thr Phe Glu
Ala Glu Tyr Asp Leu 645 650 655 Glu Arg Ala Gln Glu 660
61643PRTBacillus thuringiensis 61Met Lys Asn Ser Ile Lys Leu Ser
Glu Leu Trp Tyr Phe Asn Glu Arg 1 5 10 15 Lys Trp Arg Tyr Phe Met
Glu Ile Val Asn Asn Gln Asn Gln Cys Val 20 25 30 Pro Tyr Asn Cys
Leu Asn Asn Pro Glu Ile Glu Ile Leu Glu Gly Gly 35 40 45 Arg Ile
Ser Val Gly Asn Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr 50 55 60
Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly 65
70 75 80 Leu Ile Asp Leu Ile Trp Gly Phe Val Gly Pro Ser Gln Trp
Asp Ala 85 90 95 Phe Leu Ala Gln Val Glu Gln Leu Ile Asn Gln Arg
Ile Ala Glu Ala 100 105 110 Val Arg Asn Thr Ala Ile Gln Glu Leu Glu
Gly Met Ala Arg Val Tyr 115 120 125 Arg Thr Tyr Ala Thr Ala Phe Ala
Glu Trp Glu Lys Ala Pro Asp Asp 130 135 140 Pro Glu Leu Arg Glu Ala
Leu Arg Thr Gln Phe Thr Ala Thr Glu Thr 145 150 155 160 Tyr Ile Ser
Gly Arg Ile Ser Val Leu Lys Ile Gln Thr Phe Glu Val 165 170 175 Gln
Leu Leu Ser Val Phe Ala Gln Ala Ala Asn Leu His Leu Ser Leu 180 185
190 Leu Arg Asp Val Val Phe Phe Gly Gln Arg Trp Gly Phe Ser Thr Thr
195 200 205 Thr Val Asn Asn Tyr Tyr Asn Asp Leu Thr Glu Gly Ile Ser
Thr Tyr 210 215 220 Thr Asp Tyr Ala Val Arg Trp Tyr Asn Thr Gly Leu
Glu Arg Val Trp 225 230 235 240 Gly Pro Asp Ser Arg Asp Trp Val Arg
Tyr Asn Gln Phe Arg Arg Glu 245 250 255 Leu Thr Leu Thr
Val Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr Asp 260 265 270 Ser Arg
Arg Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile 275 280 285
Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser 290
295 300 Ala Gln Gly Ile Glu Arg Ser Ile Arg Ser Pro His Leu Met Asp
Ile 305 310 315 320 Leu Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg
Gly Tyr Tyr Tyr 325 330 335 Trp Ser Gly His Gln Ile Met Ala Ser Pro
Val Gly Phe Ser Gly Pro 340 345 350 Glu Phe Thr Phe Pro Leu Tyr Gly
Thr Met Gly Asn Ala Ala Pro Gln 355 360 365 Gln Arg Ile Val Ala Gln
Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser 370 375 380 Ser Thr Phe Tyr
Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln 385 390 395 400 Leu
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn 405 410
415 Leu Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp
420 425 430 Glu Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly
Phe Ser 435 440 445 His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly
Ser Ser Ser Ser 450 455 460 Val Ser Ile Ile Arg Ala Pro Met Phe Ser
Trp Ile His Arg Ser Ala 465 470 475 480 Glu Phe Asn Asn Ile Ile Ala
Ser Asp Ser Ile Thr Gln Ile Pro Ala 485 490 495 Val Lys Gly Asn Phe
Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly 500 505 510 Phe Thr Gly
Gly Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile 515 520 525 Gln
Asn Arg Gly Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser 530 535
540 Thr Arg Tyr Arg Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His
545 550 555 560 Leu Asn Val Asn Trp Gly Asn Ser Ser Ile Phe Ser Asn
Thr Val Pro 565 570 575 Ala Thr Ala Thr Ser Leu Asp Asn Leu Gln Ser
Ser Asp Phe Gly Tyr 580 585 590 Phe Glu Ser Ala Asn Ala Phe Thr Ser
Ser Leu Gly Asn Ile Val Gly 595 600 605 Val Arg Asn Phe Ser Gly Thr
Ala Gly Val Ile Ile Asp Arg Phe Glu 610 615 620 Phe Ile Pro Val Thr
Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg 625 630 635 640 Ala Gln
Lys
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