U.S. patent application number 12/496070 was filed with the patent office on 2010-07-01 for chimeric cryle delta endotoxin and methods of controlling insects.
This patent application is currently assigned to Council of Scientific & Industrial Research, an Indian corporation. Invention is credited to Rakesh Tuli.
Application Number | 20100168387 12/496070 |
Document ID | / |
Family ID | 35449420 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100168387 |
Kind Code |
A1 |
Tuli; Rakesh |
July 1, 2010 |
Chimeric CrylE Delta Endotoxin and Methods of Controlling
Insects
Abstract
The present invention relates to a chimeric .delta. endotoxin
protein Cry 1E of SEQ ID No. 1 with extraordinarily high
insecticidal property and a chimera gene of SEQ ID No. 2 encoding
the said chimeric protein, and also a method of treating insect
infested plants using said chimera protein.
Inventors: |
Tuli; Rakesh; (Lucknow,
IN) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Council of Scientific &
Industrial Research, an Indian corporation
|
Family ID: |
35449420 |
Appl. No.: |
12/496070 |
Filed: |
July 1, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11062225 |
Feb 18, 2005 |
7557186 |
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12496070 |
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10107581 |
Mar 27, 2002 |
7053266 |
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11062225 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C12N 15/8286 20130101;
Y02A 40/146 20180101; Y02A 40/162 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/325 20060101
C07K014/325 |
Claims
1-41. (canceled)
42. A chimeric .delta.-endotoxin protein comprising an amino acid
sequence that is identical to amino acids 530-641 of SEQ ID NO:1,
said chimeric protein having greater toxicity than Cry1C as
determined by EC.sub.50 in a challenge to neonatal Spodoptera
litura larvae.
43. A chimeric .delta.-endotoxin protein comprising an amino acid
sequence that is at least 95% identical to amino acids 1-641 of SEQ
ID NO:1, said protein having a sequence identical to amino acids
530-641 of SEQ ID NO:1, said chimeric protein having greater
toxicity than Cry1C as determined by EC.sub.50 in a challenge to
neonatal Spodoptera litura larvae.
44. The protein as claimed in claim 42 or 43, wherein said chimeric
protein is stable at temperature ranging between 4-35.degree. C.
Description
CLAIM OF PRIORITY
[0001] This application is a continuation (and claims the benefit
of priority under 35U.S.C. .sctn.120) of U.S. application Ser. No.
11/062,225, filed Feb. 18, 2005, which application is a divisional
(and claims the benefit of priority under 35 U.S.C. .sctn.120) of
U.S. application Ser. No. 10/107,581, filed Mar. 27, 2002. The
disclosure of the prior applications are considered part of (and
are incorporated by reference in) the disclosure of this
application.
FIELD OF THE PRESENT INVENTION
[0002] The present invention relates to a chimeric .delta.
endotoxin protein Cry 1E of SEQ ID No. 1 with extraordinarily high
insecticidal property and a chimera gene of SEQ ID No. 2 encoding
the said chimeric protein, and also a method of treating insect
infested plants using said chimera protein.
BACKGROUND OF THE INVENTION
[0003] Damage due to insects costs billions of dollars annually in
form of crop losses and in the expense of keeping these pests under
control. The losses caused by pests in agricultural production
environments include decrease in crop yield, poor crop quality,
increased harvesting costs, and loss to health and environment.
[0004] Reference may be made to Hofte H. and Whiteley H. R., 1989,
"Insecticidal crystal protein of Bacillus thuringiensis",
Microbiol. Rev. 53: 242-255, wherein Bacillus thuringiensis (B.t.)
is a ubiquitous gram-positive spore-forming soil bacterium, known
for its ability to produce parasporal crystalline inclusions during
sporulation. These inclusions consist of proteins known as crystal
proteins or Cry proteins or .delta.-endotoxins, which exhibit
insecticidal activity, particularly against larvae of insect
species in orders lepidoptera, diptera and coleoptera. Proteins
with toxicity to insects of orders hymenoptera, homoptera,
orthoptera, mallophaga; nematodes; mites and protozoans have also
been mentioned in literature (Feitelson J. S., 1993, "The Bacillus
thuringiensis family tree", 63-71, In L. kim ed. Advanced
engineered pesticides. Marcel Dekker. Inc., New York., N.Y. and
Feitelson et al., 1992, "Bacillus thuringiensis: insects and
beyond", Bio/Tech. 10: 271-275; may be sited for this). Several
strains of Bacillus thuringiensis (B.t.) have been identified with
different host spectra and classified into different subspecies or
serotypes on the basis of flagellar antigens. Pasteur Institute,
France has catalogued 55 different flagellar serotypes and 8
non-flagellated biotypes. The reference may be made to Schnepf et
al., 1998, "Bacillus thuringiensis and its pesticidal crystal
proteins", Microbiol. Mol. Biol. Riv. 62: 775-806, wherein several
B.t. toxin-coding genes have been cloned, sequenced, characterised
and recombinant DNA-based products to have been produced and
approved for commercial use. Through the employment of genetic
engineering techniques, new approaches have been developed for
delivering these B.t. toxins to agricultural environments,
including the use of the genetically engineered crops and the
stabilised intact microbial cells as .delta.-endotoxin delivery
vehicles (Gaertner, F. H., Kim L., 1988, TIBTECH 6: 54-57). Thus,
.delta.-endotoxin genes coding for proteins targeted to kill hosts,
especially pests and insects that cause economic losses are
becoming commercially valuable.
[0005] Commercial use of B.t. pesticides in a given crop
environment is limited because a given .delta.-endotoxin shows
toxicity to a narrow range of target pests. Preparations of the
spores and crystals of B. thuringiensis subsp. kurstaki have been
used for many years as commercial insecticides against lepidopteran
pests. For example, B. thuringiensis var. kurstaki HD-1 produces
several .delta.-endotoxins, and is therefore toxic to a relatively
broader range of lepidopteran insects. However, formulations based
on the known .delta.-endotoxins, including B.t.k. HD-1 are not
effective against some of the important crop pests, like Spodoptera
sp. that also belong to order lepidoptera. Other species of B.t.,
namely israelensis and tenebrionis have been used commercially to
control certain insects of the orders diptera and coleoptera,
respectively (Gaertner, F. H., 1989, "Cellular Delivery Systems for
Insecticidal Proteins: Living and Non-Living Microorganisms," in
Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed.,
Taylor and Francis, New York and London, 1990, pp. 245-255; Couch
T. L., 1980, "Mosquito Pathogenicity of Bacillus thuringiensis var.
israelensis", Development in Industrial Microbiology 22: 61-76 and
Beegle C. C., 1978, "Use of Entomogenous Bacteria in
Agroecosystems", "Developments in Industrial Microbiology 20:
97-104; may be sited for this). Kreig et. al. (1983) in Z. ang.
Ent. 96: 500-508, describe Bacillus thuringiensis var. tenebrionis,
which is reportedly active against two beetles in the order
Coleoptera i.e., Colorado potato beetles, Leptinotarsa decemlineata
and Agelastica alni.
[0006] Reference may be made to Crickmore et. al., 1998, "Revision
in the nomenclature for the Bacillus thuringiensis pesticidal
crystal proteins", Microbiol. Mol. Biol. Rev. 62: 807-813, wherein
crystal protein genes are classified into 22 classes, primarily on
the basis of amino acid sequence homology. The cloning and
expression of a B.t. crystal protein gene in Escherichia coli has
been described in several cases in the published literature
(Schnepf et al., 1981, "Cloning and expression of the Bacillus
thuringiensis crystal protein gene in Escherichia coli" may be
cited for this). U.S. Pat. Nos. 4,448,885 and 4,467,036 disclose
the expression of B.t crystal protein in E. coli. U.S. Pat. Nos.
4,797,276 and 4,853,331 disclose B. thuringiensis strain
tenebrionis, which can be used to control coleopteran pests in
various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins
having activity against dipterans. U.S. Pat. No. 4,849,217
discloses B.t. isolates, which have activity against the alfalfa
weevil. U.S. Pat. No. 5,208,017 discloses coelopteran-active
Bacillus thuringiensis isolates. U.S. Pat. Nos. 5,151,363 and
4,948,734 disclose certain isolates of B.t., which have activity
against nematodes. Extensive research and resources are being spent
to discover new B.t. isolates and their uses. As of now, the
discovery of new B.t. isolates and new uses of the known B.t.
isolates remains an empirical, unpredictable art. Several
laboratories all over the world are trying to isolate new
.delta.-endotoxin genes from B. thuringiensis for different host
range and mechanism of action.
[0007] Bulla et al., 1980, "Ultrastructure, physiology and
biochemistry of Bacillus thuringiensis". CRC Crit. Rev. Microbiol.
8: 147-204 and Grochulski et al., 1995, "Bacillus thuringiensis
CryIA(a) insecticidal toxin: crystal structure and channel
formation", J. Mol. Biol, 254: 447-464; have reported that majority
of B.t. insecticidal crystal proteins are synthesised in natural
form as protoxins (molecular weight 130-140 kDa), which form
parasporal inclusions by virtue of hydrophobic interactions,
hydrogen bondings and disulfide bridges. The protoxins, which are
not toxic to insect larvae, are composed of two segments the
--N-terminal half and C-terminal half. The protoxins are converted
into functionally active toxins (60-70 kDa) in insect mid gut
following their site-specific cleavage by proteases at alkaline pH.
Such proteolytically processed, truncated .delta.-endotoxins bind
to specific receptors in insect mid-gut and cause mortality by
making pores in the epithelial membrane (the references, Bietlot et
al., 1989, "Facile preparation and characterization of the toxin
from Bacillus thuringiensis var. kustaki", Biochem. J., 260: 87-91;
Choma et al., 1990, "Unusual proteolysis of the protoxin and toxin
from Bacillus thuringiensis: structural implications", Eur. J.
Biochem. 189: 523-27; and Hofte et al., 1986, "Structural and
functional analysis of a cloned delta endotoxin of. Bacillus
thuringiensis berliner 1715", Eur. J. Biochem. 161: 273-280; may be
cited for this). The protease-resistant active toxin corresponds to
N-terminal half of the protoxin molecule. The other segment
corresponding to C-terminal half is believed to be required for the
formation of highly stable crystals. During the proteolytic
processing, a small polypeptide comprising about 25-30 amino acid
residues is removed from N-terminal of the protoxin.
[0008] The crystal structure of the core toxic segment of CryIAa
and Cry3Aa .delta.-endotoxins are known (Grochulsky et al., 1995,
"Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal
structure and channel formation", J. Mol. Biol. 254: 447-464 and Li
et al., 1991, "Crystal structure of insecticidal .delta.-endotoxin
from Bacillus thuringiensis at 2.5 .ANG. resolution", Nature. 353:
815-821) and their three-dimensional structures are superimposable.
Reasonably conserved polypeptide domains suggest that related
toxins have similar topological structure. These are globular
molecules composed of 3 distinct structural domains connected by
small peptide linkers. There are no crossovers of the polypeptide
chains between the domains. Domain I consists of 7 .alpha. helical
structures. Domain II consists of three anti-parallel .beta.-sheets
and two short .alpha.-helices. Domain III is a .beta.-sandwich of
two anti-parallel highly twisted .beta.-sheets. Domains II and III
are located on the side where they face helix .alpha.7 of domain 1.
These domains are closely packed by virtue of numerous van der Wall
forces, hydrogen bonds and electrostatic interactions (salt
bridges) between the domains.
[0009] One of the major reasons for narrow host range of
.delta.-endotoxins is that these proteins need specific receptors
in the insect gut in order to make pores and cause toxicity. Since
a given .delta.-endotoxin exhibits toxicity to a very narrow range
of insects, it is desirable to engineer these proteins for
modifying their receptor recognition in larval midgut, to widen
host range and to improve toxicity. Two approaches have been
followed for this purpose--first, the development of chimeric (or
hybrid) genes by exchanging functional domains of the proteins and
secondly, the development of improved .delta.-endotoxinproteins by
site directed mutagenesis. References may be made to U.S. Pat. Nos.
5,128,130 and 5,055,294 wherein hybrid B.t. crystal proteins have
been constructed, which exhibit increased toxicity and display an
expanded host range to the target pests.
[0010] The reference may be made to Honee et al., 1990, "A
translation fusion product of two different insecticidal crystal
protein gene of Bacillus thuringiensis exhibits an enlarged
insecticidal spectrum" Appl. Environ. Microbiol. 56: 823-825,
wherein translational fusion of two cry genes (cry1Ab and cry1Ca)
has been made. The resulting hybrid protein had wider toxicity
spectrum that overlapped those of the two contributing parental
crystal proteins. However, the drawback is that the activity of the
chimeric toxin did not increased over any of the parental toxins
towards the target insect pests. In spite of poor toxicity, fusion
gene was expressed in tobacco after partial modification, which
conferred only partial protection to transgenic plants against a
broader range of insects, including Spodoptera exigua, Heliothis
virescens and Manduca sexta (the reference, van der Salm et al.,
1994, "insect resistance of transgenic plants that express modified
Bacillus thuringiensis cryIAb and cryIC genes: a resistance
management strategy", Plant Mol. Biol. 26: 51-59, may be cited for
this).
[0011] The reference may be made to Honee et at, 1991, "The
C-terminal domain of the toxic fragment of Bacillus thuringiensis
crystal protein determines receptor binding", Mol. Microbiol. 5:
2799-2806, wherein 11 chimeric genes have been constructed using
cry1Ab and cry1Ca as parent genes by exchanging functional domains.
The draw back is that only two chimeric proteins, in which
pore-forming domains had been exchanged, exhibited insecticidal
activity. However, the efficacy of the toxin chimeric proteins was
lower than the parental proteins. Other hybrid proteins were
non-toxic.
[0012] Masson et al., 1992, "Insecticidal properties of a crystal
protein gene product isolated from Bacillus thuringiensis subsp.
kenyae", Appl. Environ. Microbiol. 58: 2, 642-646, reported that
one of the Cry1 .delta.-endotoxins, namely Cry1Ea does not exhibit
toxicity against Spodoptera larvae. Further, the reference may be
made to Bosch et al., 1994, "Recombinant Bacillus thuringiensis
Crystal protein with new properties: possibilities for resistance
management", Bio/Tech 12: 915-918, wherein many chimeric genes have
been developed following in vivo recombination of cry1Ca and cry1Ea
genes. The .delta.-endotoxin expressed from one of the chimeric
genes, which consisted of domain and II of Cry1Ea and domain III of
Cry1Ca protein exhibited larvicidal activity. The transfer of
domain III of Cry1Ca to Cry1Ea protein gave an insecticidal
protein. However, the chimeric toxin was not an improved toxin over
the Cry1Ca, which is best-reported toxin to Spodoptera sp. Another
chimeric toxin exhibited very poor toxicity. The remaining chimeric
toxins were either unstable or non-toxic.
[0013] Reference may be cited as Rang et al., 1999, "Interaction
between functional domain of Bacillus thuringiensis insecticidal
crystal protein", Appl Environ Microbial, 65, 7: 2918-25, wherein
many chimeric genes have been developed by exchanging the regions
coding for either domain 1 or domain III among Cry1Ab, Cry1Ac,
Cry1Ca and Cry1Ea .delta.-endotoxins and checked their stability in
E. coli and plasma membrane permeability of Sf9 cells. A chimeric
toxin (consisting of domains I and II of Cry1Ca and domain III of
Cry1Ab) was more toxic than the parental toxins. Exchange of domain
III of Cry1Ab with that of Cry1Ca made the chimeric protein more
active than the Cry1Ca protein. Proteins with the exchange of other
domains were either unstable or less toxic than the parent
proteins. However, the toxicity of the chimeric protein to insect
larvae was not tested. Pore formation in insect cell line was
compared but that cannot be correlated with the insecticidal
activity of the .delta.-endotoxin.
[0014] Reference may be made to Chandra et al., 1999, "Amino acid
substitution in alpha-helix 7 of Cry1Ac .delta.-endotoxin of
Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa
armigera Hubner", FEBS Lett. 458: 175-179; wherein a hydrophobic
motif in the C-terminal end of the fragment B of diphtheria toxin
was found to be homologous to helix .alpha.7 of .delta.-endotoxins.
Upon substitution of helix .alpha.7 of Cry1Ac protein by this
polypeptide, the chimeric protein exhibited 7-8 fold enhancement in
toxicity towards Helicoverpa armigera. The increased toxicity was
due to higher pore forming ability.
[0015] These examples establish the potential of protein
engineering for the improvement of native toxins, to develop
commercially useful .delta.-endotoxins.
[0016] Most of the lepidopteran pests are polyphagous in nature.
Spodoptera is a common lepidopteran insect and its 5 species
(litura, littoralis, exigua, frugiperda and exempia) are found
worldwide. Spodoptera littoralis (the Egyptian cotton leaf worm,
CLW) is a major pest of cotton and other crops of agronomical
importance in Europe (the reference Mazier et al., 1997, "The cryIC
gene from Bacillus thuringiensis provides protection against
Spodoptera littoralis in young transgenic plants", Plant Sci. 127:
179-190, 190, may be cited for this). It is a notorious pest of
cotton, groundnut, chilli, pulses and several vegetable crops,
especially in warm and humid regions, as the southern parts of
India. High fecundity, short life cycle, destructive feeding habits
and often-reported emergence of resistance to chemical insecticides
have made the control of Spodoptera an increasing agricultural
problem. Reference may be made to Bai et al., 1993, "Activity of
insecticidal proteins and strains of Bacillus thuringiensis against
Spodoptera exempla (Walker)" J. Inverteb. Pathol. 62: 211-215,
wherein it is discussed that the young larvae are susceptible to
certain .delta.-endotoxins, but the larvae beyond 2.sup.nd instar
display considerable tolerance. This has been attributed to the
high content of alkaline proteases in the gut juice (the reference
Keller et al., 1996, "Digestion of .delta.-endotoxin by gut
proteases may explain reduced sensitivity of advanced instar larvae
of Spodoptera littoralis to CryIC", Insect Biochem. Mol. Biol. 26:
365-373, may be cites for this).
[0017] Four different .delta.-endotoxins have been reported to
cause low level of mortality to the Spodoptera sp. Of these, Cry1Ca
is the most effective toxin. The plants expressing Cry1Ca at a high
level caused mortality and hence conferred protection against early
instar larvae (the references Mazier et al., 1997, "The cryIC gene
from Bacillus thuringiensis provides protection against Spodoptera
littoralis in young transgenic plants". Plant Sci. 127: 179-190 and
Strizhov et al., 1996, "A synthetic cry1C gene, encoding a Bacillus
thuringiensis .delta.-endotoxin, confers Spodoptera resistance in
alfalfa and tobacco" Proc. Natl. Acad. Sci. USA. 93: 15012-15017
may be cited for this). However, complete protection against
Spodoptera has not been reported in any case. The larvae in
advanced developmental stages are not killed at moderate levels of
the known 6-endotoxins. Hence, transgenic plants expressing Cry1Ca
are not as effective as desirable in protection against Spodoptera.
Other genes, like cry1Cb, cry1Ea and cry1F have not been employed
for the development of transgenic plants against Spodoptera because
of their comparatively low toxicity. Cry1Cb .delta.-endotoxin is
5-fold less toxic than Cry1Ca. The toxicity of Cry1Ea
.delta.-endotoxin is very low and is disputed in certain reports
(Masson et al., 1992 and Bosch et al., 1994 may be cited for this).
Cry1F exhibits mild toxicity to Spodoptera larvae (Chambers et al.,
1991 may be cited for this).
[0018] Reference may be made to Kalman et al., 1993, "Cloning of a
novel cryIC-type gene from a strain of Bacillus thuringiensis
subsp. Galleriae", Appl. Environ. Microbiol. 59:4:1131-1137,
wherein Cry1Cb .delta.-endotoxin is reported to be 5-fold less
toxic than Cry1Ca. First two domains of these proteins are highly
homologous (92% identical). A major difference is observed in
domain III that exhibits only 48% homology. Higher toxicity
(5-fold) of Cry1Ca over Cry1Cb .delta.-endotoxin suggested us that
domain III of Cry1Ca might have an important role in its efficacy.
The toxicity of Cry1Ea .delta.-endotoxin is very poor as it binds
to the receptor very weakly in the midgut of Spodoptera exigua but
the exchange of Domain III of Cry1Ca with Cry1Ea, made the latter
toxic. This suggests the role of Domain III of Cry1C protein in
receptor binding in the midgut of Spodoptera (the reference Bosch
et. al., 1994, "Recombinant Bacillus thuringiensis Crystal protein
with new properties: possibilities for resistance management".
Bio/Tech 12: 915-918, may be cited for this). In this publication,
Bosch et al. (1994) established the advantage of hybrid toxin as it
binds to a receptor where Cry1Ca does not bind. However, the
toxicity of both, the native Cry1Ca and the hybrid Cry1Ea was
comparable. They filed a patent (U.S. Pat. No. 5,736,131) in which
1.9-fold improvement in the toxicity towards Spodoptera exigua was
claimed. The difference in results in the publication that reports
no enhancement in toxicity and in the patent that claims 1.9 fold
improved toxicity, makes the overall picture unclear.
[0019] Plant genetic engineering technology has made significant
progress during the last 10 years. It has become possible to stably
introduce foreign genes into plants. This has provided exciting
opportunities for modern agriculture. Derivatives of the Ti-plasmid
of the plant pathogen, Agrobacterium tumefaciens, have proven to be
efficient and highly versatile vehicles for the introduction of
foreign genes into plant tissue. In addition, a variety of methods
to deliver DNA, such as electroporation, microinjection,
pollen-mediated gene transfer and particle gun technology, have
been developed for the same purpose.
[0020] The major aim of plant transformation by genetic engineering
has been crop improvement. A substantial effort has been made for
engineering the plants for useful traits such as insect-resistance.
In this respect, progress in engineering insect resistance in
transgenic plants has been achieved through the use of genes,
encoding .delta.-endotoxins, from B. thuringiensis strains. Since
.delta.-endotoxins possess a specific insecticidal spectrum and
display no toxicity towards other non-host animals and humans,
these are highly suited for developing commercially useful plants.
No other protein is known which shows as high toxicity as (at ppm
levels) and is still as safe to non-target organisms as the
.delta.-endotoxins.
[0021] The feasibility of generating insect-resistant transgenic
crops expressing 8-endotoxins and their success in commercial
agriculture has been demonstrated. (References may be made to Vaeck
et al., 1987, "Transgenic plants protected from insect attack",
Nature. 328: 33-37; Fischoff et al., 1987, "Insect tolerant
transgenic tomato plants", Bio/Tech. 5: 807-813''; Barton et al.,
1987, "Bacillus thuringiensis .delta.-endotoxin expressed in
transgenic Nicotiana tabaccum provides resistance to lepidopteran
insects", Plant Physiol. 85: 1103-1109 may be made for this).
Transgenic plants offer an attractive alternative to insect control
in agriculture, which is at the same time safe, environment
friendly and cost-effective. Successful insect control has been
observed under field conditions (Reference may be made to Delannay
et al., 1989; Meeusen and Warren, 1989).
[0022] A reference may be cited to Von Tersch et al. 1991,
"Insecticidal toxins from Bacillus thuringiensis subsp kanyae: Gene
cloning and characterization and comparison with B. thuringiensis
subsp kurstaki Cry1A(c) toxin" in Appl and Environ Microbial, 57:
2: 349-58, wherein two variants of cry1Ac were isolated from two
different strains. Their amino acid composition was different at 7
positions. Both the .delta.-endotoxins were expressed in E. coli
and toxicity experiment was conducted. The two toxins did not
exhibit any difference in efficacy towards target pests.
[0023] In another study (Schnepf et al., 1998, "Bacillus
thuringiensis and its pesticidal crystal proteins", 62: 3,
775-806), amino acid residues GYY of Cry1Ac .delta.-endotoxin at
position 312 to 314 were altered to replace these with ASY, GSY and
GFS. No difference in toxicity of the three proteins was
noticed.
[0024] There are two natural variants of Cry1C .delta.-endotoxin
namely Cry1Ca and Cry1Cb. These proteins show 81% (Schnepf et al.,
1998, "Bacillus thuringiensis and its pesticidal crystal proteins",
62: 3, 775-806) amino acid sequence identity. Despite this
difference, both the toxins are toxic to their target pest, though
there is some difference in the level of toxicity. Their host range
is also same. (Kalman et al., 1993. "Cloning of a noval cry1C type
gene from a strain of Bacillus thuringiensis subsp galleriae" Appl.
Environ Microbial 59: 4: 1131-37)
OBJECTS OF THE PRESENT INVENTION
[0025] The main object of the present invention is to develop a
chimeric .delta. endotoxin protein.
[0026] Another main object of the present invention is to develop a
chimera gene coding for a chimeric .delta. endotoxin protein.
[0027] Yet another object of the present invention is to develop a
method of developing said chimeric protein.
[0028] Still another object of the present invention is to develop
a method of overexpressing said chimeric protein in a suitable
microbe.
[0029] Still another object of the present invention is to develop
a method of treating insect infested plants using said chimeric
protein.
[0030] Further object of the present invention is to develop an
insecticide for multidrug resistant insects.
[0031] Another object of the present invention is to develop an
effective insecticide.
[0032] Yet another object of the present invention is to develop an
insecticide having no adverse effect on the plants.
[0033] Still another object of the present invention is to develop
an insecticide with about 100% insecticidal activity.
SUMMARY OF THE PRESENT INVENTION
[0034] The present invention relates to a chimeric .delta.
endotoxin protein Cry 1E of SEQ ID No. 1 with extraordinarily high
insecticidal property and a chimera gene of SEQ ID No. 2 to
encoding the said chimeric protein, and also a method of treating
insect infested plants using said chimera protein.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
[0035] Accordingly, the present invention relates to a chimeric
.delta. endotoxin protein Cry 1E of SEQ ID No. 1 with
extraordinarily high insecticidal property and a chimera gene of
SEQ ID No. 2 encoding the said chimeric protein, and also a method
of treating insect infested plants using said chimera protein.
[0036] In one embodiment of the present invention, a chimeric
.delta. endotoxin protein Cry1E of SEQ ID No. 1 and other proteins
of 75% and above homology in the sequence.
[0037] In another embodiment of the present invention, a chimeric
.delta. endotoxin protein Cry1E of SEQ ID No. 1.
[0038] In yet another embodiment of the present invention, wherein
said protein is of length 641 amino acid residues.
[0039] In still another embodiment of the present invention,
wherein said chimeric protein is designed from .delta. endotoxins
Cry1Ea and Cry1Ca of Bacillus thuringiensis.
[0040] In still another embodiment of the present invention,
wherein said chimeric protein of length of 641 residues is
consisting of residues 1 to 529 from endotoxin Cry1Ea of same
position, residues 530 to 599 from Cry1Ca of position 533 to 602,
residues 600 to 616 from Cry1Ea of position 588 to 604 and residues
617 to 641 of a synthetic polypeptide.
[0041] In still another embodiment of the present invention,
wherein peptide domain from 530 to 587 of Cry1Ea can be replaced
with that of any other .delta. endotoxin.
[0042] In still another embodiment of the present invention,
wherein last 25 amino acid residues improve the stability against
the proteases of plants.
[0043] In still another embodiment of the present invention,
wherein said chimeric protein is stable at temperature ranging
between 4-35.degree. C.
[0044] In further embodiment of the present invention, A chimera
gene of SEQ ID No. 2.
[0045] In another embodiment of the present invention, wherein said
chimera encodes chimeric protein of SEQ ID No. 1.
[0046] In still another embodiment of the present invention,
wherein said chimera is of length 1990 base pairs (bp).
[0047] In still another embodiment of the present invention,
wherein said chimera is a 1.99-kb double stranded DNA.
[0048] In still another embodiment of the present invention,
wherein said chimera contains plant preferred codon distributed
evenly to facilitate efficient translation.
[0049] In still another embodiment of the present invention,
wherein said chimera contains plant preferred translation
initiation codon of ATGGCT at 5' extreme.
[0050] In still another embodiment of the present invention,
wherein said chimera contains plant preferred translation
termination codon of TAATGA.
[0051] In still another embodiment of the present invention,
wherein said chimera contains 33 restriction sites distributed
uniformly throughout the length of the gene at a distance of about
40-80 bp.
[0052] In still another embodiment of the present invention,
wherein restriction sites are enzymes selected from a group
comprising Hind III, EcoRI, and BamHI.
[0053] In still another embodiment of the present invention,
wherein said chimera is divided into 58 overlapping
oligonucleotides of length 40 to 65 by each located at a distance
of 6 to 26 base pairs. (bp)
[0054] In still another embodiment of the present invention,
wherein said chimera contains said overlapping nucleotides with an
overlap of 13 to 18 nucleotides with the immediately adjacent
oligonucleotides on the complementary strain.
[0055] In still another embodiment of the present invention,
wherein said chimera has T.sub.m value ranging between 44 to
55.degree. C.
[0056] In further embodiment of the present invention, a method of
overexpressing insecticidal chimeric protein Cry1E in microbes.
[0057] In another embodiment of the present invention, cloning gene
Cry1E of SEQ ID No. 2 encoding said chimeric protein in a
vector.
[0058] In still another embodiment of the present invention,
transforming microbe with said cloned vector.
[0059] In still another embodiment of the present invention,
overexpressing said chimeric protein into said microbe.
[0060] In still another embodiment of the present invention,
wherein said chimera is expressed into a microbe selected from a
group comprising bacteria, algae, and fungi.
[0061] In still another embodiment of the present invention,
wherein restriction enzymes for said cloning are selected from a
group comprising Hind III, EcoRI, Ncol, Mfe I and BamHI.
[0062] In still another embodiment of the present invention,
wherein inducing overexpression of said protein by using
isopropylthiogalactoside (IPTG).
[0063] In still another embodiment of the present invention,
wherein overexpressing said protein at 15.degree. C. to avoid
mis-folding of said proteins.
[0064] In still another embodiment of the present invention,
wherein said vectors are selected from a group comprising Plasmids,
viral DNA, and cosmids.
[0065] In still another embodiment of the present invention,
wherein expression of chimera in the microbe is confirmed by
RT-PCR, western Analysis, and ELISA.
[0066] In still another embodiment of the present invention,
wherein presence of chimera in the microbe is confirmed by PCR and
southern Analysis.
[0067] In further embodiment of the present invention, a method of
treating plants infected with insects using said insecticidal
chimeric protein.
[0068] In another embodiment of the present invention,
incorporating gene encoding chimera protein Cry1E into plant
infected with insects.
[0069] In yet another embodiment of the present invention, exposing
transgenic plant to insects.
[0070] In still another embodiment of the present invention,
determining insecticidal activity of said transgenic plants.
[0071] In still another embodiment of the present invention,
wherein insect pests are selected from a group comprising
spodoptera sp., and Helicoverpa sp.
[0072] In still another embodiment of the present invention,
wherein plants are selected from a group comprising tobacco,
cotton, chickpea, pegeonpea, groundnut, cauliflower, cabbage,
chilli, and capsicum.
[0073] In still another embodiment of the present invention,
wherein restriction enzymes for said cloning are selected from a
group comprising Hind III, EcoRI, Ncol, and BamHI.
[0074] In still another embodiment of the present invention,
wherein chimeric protein shows high degree of expression in plants
by having about 0.5% of total soluble protein of plants.
[0075] In still another embodiment of the present invention,
wherein said chimeric protein is stable in said transgenic
plant.
[0076] In still another embodiment of the present invention,
wherein insects exposed to said chimeric protein show weight loss
before death.
[0077] In still another embodiment of the present invention,
wherein said chimeric protein shows insecticidal property against
insect at all developmental stages.
[0078] In still another embodiment of the present invention,
wherein said chimeric protein is multifold more potent insecticide
as compared to parental proteins.
[0079] In still another embodiment of the present invention,
wherein insecticidal activity of said chimeric protein shows
mortality of insect pests ranging between 80-100% within about 4
hours of exposure.
[0080] In still another embodiment of the present invention,
wherein insects exposed to said chimeric protein for about 1 hour
shows delayed development, infertility and disrupted
metamorphosis.
[0081] In still another embodiment of the present invention,
wherein EC.sub.50 for Helicoverpa sp. is ranging between 250-350
ng/ml of artificial diet of insects.
[0082] In still another embodiment of the present invention,
wherein EC.sub.50 of Spodoptera sp. is ranging between 25-50 ng/ml
of artificial diet.
[0083] In still another embodiment of the present invention, the
said method shows no adverse effect on the normal growth of the
transformed plants.
[0084] In further embodiment of the present invention, a novel
chimeric Bacillus thuringiensis .delta.-endotoxin is strategically
developed by replacing a polypeptide domain of a .delta.-endotoxin,
herein said Cry1Ea with the corresponding domain of other
.delta.-endotoxins and a novel polypeptide at the C-terminus
extreme. A gene is theoretically designed and chemically
synthesized to encode the novel chimeric toxin and express it at
high level in plant tissue. The gene was expressed in a microbe (E.
coli) and two dicot plants (tobacco and cotton). Efficacy of the
strategically designed .delta.-endotoxin was established in both
the systems. The toxicity of the chimeric protein to lepidopteran
insect larvae (Spodoptera and Helicoverpa) was improved as compared
to the parent proteins. The chimeric synthetic gene is commercially
valuable as it can be used to develop agronomically improved crop
plants for resistance to insect pests.
[0085] In another embodiment of the present invention, accordingly,
the present invention provides "a novel .delta.-endotoxin improved
for insecticidal activity and a gene for its high level expression
in plants" which comprises strategic designing of a novel chimeric
.delta.-endotoxin, herein said chimeric Cry1E (616 amino acid
residues), by replacing a polypeptide domain (from position 530 to
587) of Cry1Ea protein by that of Cry1Ca (from position 533 to
602), incorporation of a novel polypeptide of 25 amino acid
residues at the C-terminus extreme, theoretical designing of a gene
to code 641 amino acid residue long chimeric .delta.-endotoxin at a
high level in plants, designing and chemical synthesis of
oligonucleotides representing theoretically designed gene, assembly
of oligonucleotides into double stranded DNA, cloning and sequence
analysis of cloned synthetic DNA, construction of vectors for the
expression of chimeric gene in E. coli and plants, expression of
synthetic chimeric gene in E. coli, comparison of the toxicity of
the chimeric protein with the parental proteins against Spodoptera
litura and Helicoverpa armigera, transformation of tobacco with the
chimeric gene, high level expression of the engineered protein in
transgenic plants, purification of chimeric .delta.-endotoxin
protein from transgenic plants and confirmation of its efficacy on
larvae of Spodoptera litura and Helicoverpa armigera, evaluation of
the potential of the chimeric toxin expressed in transgenic plants
for protection against target insect pests.
[0086] In an embodiment of the present invention, a naturally
occurring .delta.-endotoxin, namely Cry1Ea has been strategically
designed and modified accordingly to make it biologically active
against larvae of insect pests, like Spodoptera sp.
[0087] In another embodiment of the present invention, 616 amino
acid residue long chimeric .delta.-endotoxin, herein said chimeric
Cry1E, was theoretically designed by replacing a peptide domain of
Cry1Ea from position 530 to 587 by that of Cry1Ca from position 533
to 602. Further, a novel polypeptide of 25 amino acid residues was
introduced at the C-terminus extreme of .delta.-endotoxin.
[0088] In yet another embodiment of the present invention, two
peptide domains from positions 530 to 545 and 578 to 587 of Cry1Ea
can be replaced by those of Cry1Ca from 533 to 555 and 588 to 602.
Such chimeric toxin may also perform similar (or equivalent)
biological activity.
[0089] In still another embodiment of the present invention, the
peptide domain from 530 to 587 of Cry1Ea can be replaced by that of
other .delta.-endotoxins.
[0090] In still another embodiment of the present invention, a
polypeptide of 25 amino acid residues is introduced at the
C-terminus of .delta.-endotoxin. This polypeptide improved the
stability of .delta.-endotoxin against the proteases of plant. This
might have improved the stability of the .delta.-endotoxin insect
mid gut.
[0091] In still another embodiment of the present invention, 25
amino acid residues long polypeptide may be included at the
C-terminus of other .delta.-endotoxins, such toxins may become
stable against different kind of proteases.
[0092] In still another embodiment of present invention, 25 amino
acid residues long polypeptide may be incorporated at the
C-terminus of any recombinant protein for their stability against
proteases.
[0093] In still another embodiment of the present invention, a
1.99-kb double stranded DNA was theoretically designed to encode
the chimeric protein. Plant preferred codons were used to encode
amino acids of the chimeric toxin protein to facilitate high-level
expression of the gene in plants.
[0094] There are 6 variants of Cry1Aa .delta.-endotoxin namely
Cry1Aa1 to Cry1Aa6 given in EMBL database. Clustal analysis for
amino acid sequence of these .delta.-endotoxin proteins is shown
below. At three positions (77, 148 and 302), the amino acid
residues are different. The variant positions are shown below in
bold letters. However, all six genes have been deployed in toxicity
experiments by different laboratories. The efficacy of all six
variants in their toxicity towards insects is comparable.
TABLE-US-00001 CRY1AA1
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA4
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA5
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA6
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA2
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA3
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLL 50 SEF CRY1AA1
VPGAGFVLGLVDIIWGIFGPSQWDAFPVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA4
VPGAGFVLGLVDIIWGIFGPSQWDAFPVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA5
VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA6
VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA2
VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA3
VPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAI 100 SRL CRY1AA1
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA4
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA5
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA6
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 LAV CRY1AA2
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 FAV CRY1AA3
EGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPL 150 FAV CRY1AA1
QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA4
QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA5
QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA6
QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA2
QNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLT 200 RLI CRY1AA3
QNYQVPLLSVYVQAANLHLSVLRDVSVPGQRWGFDAATINSRYNDLT 200 RLI CRY1AA1
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA4
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA5
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA6
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA2
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA3
GNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALF 250 SNY CRY1AA1
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA4
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA5
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA6
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA2
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA3
DSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQRIEQNIRQPHLM 300 DIL CRY1AA1
NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA4
NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA5
NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA6
NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA2
NRITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA3
NSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGNAAPP 350 VLV CRY1AA1
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA4
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA5
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA6
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA2
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA3
SLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNL 400 PST CRY1AA1
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA4
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA5
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA6
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA2
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA3
IYRQRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTL 450 RAP CRY1AA1
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA4
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA5
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA6
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA2
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA3
TFSWQHRSAEFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGD 500 ILR CRY1AA1
RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA4
RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF CRY1AA5
RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550
GNF CRY1AA6 RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF
CRY1AA2 RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF
CRY1AA3 RTSPGQISTLRVNITAPLSQRYRVRIRYASTTNLQFHTSIDGRPINQ 550 GNF
CRY1AAI SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA4 SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA5 SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA6 SATMSSGSNLQSGSFRTVGFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA2 SATMSSGSNLQSGSFRTVCFTTPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA3 SATMSSGSNLQSGSFRTVCFFFPFNFSNGSSVFTLSAHVFNSGNEVY 600 IDR
CRY1AA1 IEFVPAEVT 609 (SEQ ID NO: 3) CRY1AA4 IEFVPAEVT 609 (SEQ ID
NO: 4) CRY1AA5 IEFVPAEVT 609 (SEQ ID NO: 5) CRY1AA6 IEFVPAEVT 609
(SEQ ID NO: 6) CRY1AA2 IEFVPAEVT 609 (SEQ ID NO: 7) CRY1AA3
IEFVPAEVT 609 (SEQ ID NO: 8)
[0095] A reference may be cited to Von Tersch et al. 1991,
"Insecticidal toxins from Bacillus thuringiensis subsp kanyae: Gene
cloning and characterization and comparison with B. thuringiensis
subsp kurstaki Cry1A(c) toxin" in Appl and Environ Microbial, 57:
2: 349-58, wherein two variants of cry1Ac were isolated from two
different strains. Their amino acid composition was different at 7
positions. Both the .delta.-endotoxins were expressed in E. coli
and toxicity experiment was conducted. The two toxins did not
exhibit any difference in efficacy towards target pests.
[0096] Further, this clearly states that in proteins, more
particularly in the field of endotoxins, the high homology of the
sequence is not found to make any significant difference in
activity. The above-referred example of endotoxins Cry1Aa1 to
Cry1Aa6 clearly reflect the essence of this work. In the instant
Application, the applicant has observed extraordinarily high
insecticidal activity. Further, the homology of 70% and above in
the sequence of chimeric protein Cry1E of the instant Application
is also found to show no significant change in the activity. This
means that the proteins with sequence homology of 70% and above for
chimeric protein Cry 1E are used as insecticidal agents.
[0097] In another study (Schnepf et al., 1998, "Bacillus
thuringiensis and its pesticidal crystal proteins", 62: 3,
775-806), amino acid residues GYY of Cry1Ac .delta.-endotoxin at
position 312 to 314 were altered to replace these with ASY, GSY and
GFS. No difference in toxicity of the three proteins was
noticed.
[0098] In addition, there are two natural variants of Cry1C
.delta.-endotoxin namely Cry1Ca and Cry1Cb. These proteins show 81%
(Schnepf et al., 1998, "Bacillus thuringiensis and its pesticidal
crystal proteins", 62: 3, 775-806) amino acid sequence identity.
Despite this difference, both the toxins are toxic to their target
pest, though there is some difference in the level of toxicity.
Their host range is also same. (Kalman et al., 1993. "Cloning of a
noval cry1C type gene from a strain of Bacillus thuringiensis subsp
galleriae "Appl. Environ Microbial 59: 4: 1131-37)
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0099] FIG. 1 shows represents PCR based assembly of 58 overlapping
oligonucleotides into 1.99 kb novel chimeric cry1E DNA. Lanes 2-4
represent gene assembly in different PCR conditions, the desired
DNA fragment is shown with arrow Lane 1 showing
.lamda.DNA/HindIII-EcoRI molecular weight marker.
[0100] FIG. 2 represents the restriction analysis of plasmid pPK59
having E-35-S promoter at the upstream of the novel chimeric gene.
The plasmid was digested with SalI restriction enzyme (40 by
downstream of gene). The linear plasmid was further digested with
NcoI (lane 2), NheI (lane 3), BstXI (lane 4), NruI (Lane 5) and
SacI (lane 6). Lane 1 and 7 represent .lamda.DNA/HindIII-EcoRI and
.lamda.DNA/HindIII molecular weight markers, respectively.
[0101] FIG. 3 represent map of plasmids pPK202, pPK141, pPK135 and
pPk206.
[0102] FIG. 4 represents insect bioassay with transgenic tobacco
plants. 1.sup.st instar larvae of Spodoptera litura showing 100%
mortality after 2 days of feeding on transgenic leaf expressing the
novel chimeric cry1E gene (left). Control leaf was eaten by larvae
voraciously (right).
[0103] FIG. 5 represents 3.sup.rd instar larvae of Spodoptera
litura showing 100% mortality after 2 days of feeding (left).
Transgenic plant leaf exhibited high level of protection against
larvae in contrast to the control plant leaf (right).
[0104] FIG. 6 represents 100% mortality of 5.sup.th instar larvae
of Spodoptera following 48 h of feeding on transgenic tobacco leaf
(left). During same period, the larvae fed on control plant leaf
ingested 6 leaves (right).
[0105] In still another embodiment of the present invention, 58
oligonucleotides were designed to represent 1.99-kb chimeric cry1E
DNA. All oligonucleotides were synthesised chemically and fused
enzymatically to obtain desired double stranded DNA. (Please Refer
FIG. 1)
[0106] In still another embodiment of the present invention, two
independent constructs were made, each for E. coli and plant
expression. The parental genes (cry1Ea and cry1Ca) were also
introduced in other expression vectors for their expression in E.
coli.
[0107] In still another embodiment of the present invention, all
three genes were expressed in E. coli. The efficiency of the
chimeric Cry1E against Spodoptera litura was compared to the
parental toxins (Cry1Ea and Cry1Ca). The toxicity experiments
established that the engineered toxin is several fold more toxic as
compared to the parental proteins.
[0108] In still another embodiment of the present invention, the
chimeric protein expressed in plants was shown to be also toxic to
Helicoverpa armigera, another serious insect pest. This established
the improvement in the host range of the novel chimeric toxin
designed in this study.
[0109] In still another embodiment of the present invention, the
synthetic gene was introduced in tobacco for expression of the
chimeric toxin. The transgenic plants expressed chimeric toxin and
accumulated up to 0.5% of total soluble protein. The transgenic
plants exhibited excellent protection against larvae of Spodoptera
litura and caused 100% mortality at all the developmental
stages.
[0110] In still another embodiment of the present invention, the
novel chimeric toxin was purified from total soluble protein from
the leaf of the transgenic tobacco plant and mixed in semi
synthetic diet. The toxicity experiments again established the
efficacy of the hybrid toxin.
[0111] The subject invention concerns the discovery of highly
active chimeric .delta.-endotoxins. A novel .delta.-endotoxin, 641
amino acid residues long, herein said chimeric Cry1E was
strategically designed by replacing d polypeptide domain (from
position 530 to 587) of Cry1Ea protein by that of Cry1Ca (from
position 533 to 602). In this way chimeric toxin comprises amino
acid residues 1-529 of Cry1Ea, 530 to 599 of Cry1Ca and 600 to 616
of Cry1Ea. A novel polypeptide of 25 amino acid residues was
included as the C-terminus extreme of the .delta.-endotoxin. In
other words, this polypeptide constituted amino acid residue
617-641 of the chimeric toxin. Several chimeric toxins can be
created by replacing different parts of Cry1Ea toxin with
strategically designed amino acid sequences or parts of the other
toxins. A 1.99 kb nucleotide sequence was theoretically designed to
code for the above-mentioned chimeric .delta.-endotoxin. The gene
encoding toxin protein, herein said chimeric cry1E was designed for
high-level expression in plants, by introducing plant-preferred
codons. The plant preferred codons for each amino acid were
distributed evenly to facilitate efficient translation. A
translation initiation context appropriate to gene expression in
plants (TAAACCATGGCT; (SEQ ID NO:9)) was included at 5' extreme and
two translation stop codons (signals) were introduced at the end of
the reading frame of the chimeric toxin. A total of 33 unique
restriction sites were introduced uniformly throughout the length
of the gene at a distance of 40-80 bp. BamHI and HindIII
restriction sites were created at the upstream and BamHI and EcoRI
at the downstream of the gene to facilitate its cloning. (Please
refer FIG. 2).
[0112] The translation initiation context automatically created an
NcoI site at the immediate start of the gene. The gene was divided
into 58 overlapping oligonucleotides (40 to 65 nucleotides long)
with 6 to 26 base long gaps in between (Please refer FIG. 1). Each
oligonucleotide had 13-18 nucleotide long overlap with the
immediately adjacent oligonucleotides on the complementary strand.
The complementary overlaps were designed to keep T.sub.m value
between 48-50.degree. C. The oligonucleotides were synthesised on a
DNA synthesiser (Gene Assembler Special, Pharmacia, Sweden) at 200
nmole scale and purified on denaturing urea-PAGE. All 58
oligonucleotides were assembled into 1.99 kb double-stranded DNA,
herein said chimeric cry1E gene following the ligation-free gene
synthesis method of Singh et al. (1996) and as shown in FIG. 1. The
DNA was digested with HindIII and EcoRI restriction enzymes and
cloned in pBluescriptll SK(+) (Stratagene, La Jolla, Calif.). The
plasmid was named as pPK200. The nucleotide sequence of the
synthetic DNA was confirmed by sequencing the cloned synthetic DNA
on automated DNA sequencing system (Applied Biosystems model
373A).
[0113] A cassette was constructed also for the expression of the
chimeric toxin in E. coli under the control of T7lac promoter. The
plasmid pPK200 was digested with the restriction enzymes NcoI and
BamHI and cloned in expression vector pET-19b (Novagen, Madison
Wis.). The plasmid was named as pPK206. DNA encoding Cry1Ca and
Cry1Ea toxins were amplified with polymerase chain reaction, using
suitable primers, which created NcoI and BamHI restriction sites at
the upstream and the downstream of the amplicon. The amplified
products were cloned in the pET-19b vector. The constructs having
Cry1Ca and Cry1Ea toxin coding DNA were named as pPK135 and pPK141,
respectively. E. coli BL21DE3 strain was transformed with the
constructs pPK206, pPK135 and pPK141. (Please refer FIG. 3). The
toxin proteins were expressed by induction with appropriate
concentrations of IPTG. The expression was carried out at
15.degree. C. to avoid possible mis-folding. E. coli cells were
lysed with lysozyme and sonicated to release the
.delta.-endotoxins. The toxin proteins were found in inclusion
bodies. These were solubilised in 50 mM Bicarbonate buffer (pH 9.5)
at 28.degree. C. The toxin proteins were quantified
densitometrically. Serial dilutions of the toxins were mixed in
semi-synthetic diet and the mixture was poured in the petri dishes.
Total E. coli protein served as control diet. Fifteen neonatal
larvae of Spodoptera litura were released onto the cakes of the
diet mixture in a 100-ml beaker and the mouth was covered with
muslin cloth to allow gas exchange. Each experiment was conducted
with 6 replicates. The diet was changed after every alternate day.
Bio-assay was conducted with 16/8 h photoperiod at
25.+-.0.2.degree. C. Toxicity data was recorded after 7 days of the
feeding. EC.sub.50 was determined by standard log-probit analysis.
All three proteins were tested simultaneously. The representative
results are as shown in Table 1 here below.
TABLE-US-00002 TABLE 1 .delta.-endotoxins(S) EC.sub.50 (.mu.g/ml
semisynthetic diet) Cry1Ea >108 Cry1Ca 29.48 .+-. 1.77 Chimeric
Cry1E 6.27 .+-. 0.59
[0114] The result showed that the E. coli strain expressing the
chimeric toxin was several fold more toxic over Cry1Ea and more
than four fold toxic over Cry1Ca protein. The Cry1Ea toxin protein
failed to cause any effect on the Spodoptera larvae. The result
established the successful engineering of the Cry1Ea toxin to
develop a novel protein chimeric Cry1E, which is biologically
active and an improved toxin. The engineered protein was four fold
more toxic than Cry1Ca protein, which is the best-known 8-endotoxin
against Spodoptera sp.
[0115] All three .delta.-endotoxins were also tested against 72-h
old larvae of Helicoverpa sp. Each larva was released on diet in a
separate box. 40 larvae were challenged with each concentration of
the toxin. The toxicity results show that the chimeric toxin is
also toxic to Helicoverpa. A representative result is shown in
Table 2 as here below.
TABLE-US-00003 TABLE 2 .delta.-endotoxins(S) EC.sub.50 (.mu.g/ml
semisynthetic diet) Cry1Ea >176 Cry1Ca 136.22 .+-. 8.77 Chimeric
Cry1E 26.71 .+-. 1.39
[0116] As used here, reference to the "toxin" means, the N-terminal
segment, which is responsible for the insect pesticidal activity. A
person skilled in this art can convert this toxin into a protoxin
by including a part or complete C-terminus fragment of a homologous
or hetrologous .delta.-endotoxin. Such protoxin will be a very
stable molecule inside a microbial cells for example, in E. coli,
Pseudomonas etc. and can be used in developing microbial
formulations. Such formulations can be used as pesticides.
Development of such protoxins and formulations using the novel
toxin developed by us is also within the scope of the invention
claimed by us.
[0117] The gene and toxin useful according to the subject invention
include not only 641 amino acid long toxin but also fragments of
the novel sequence, variants and mutants, which retain the
characteristic pesticidal activity of the toxin specifically
exemplified herein. As used here, the terms "variants" or
"variations" of genes refer to nucleotide sequences, which encode
the same toxins or which encode toxins having lower or equivalent
pesticidal activity. As used here, the term "equivalent pesticidal
activity" refers to toxins having similar or essentially the same
biological activity against the target pests as the claimed
toxins.
[0118] It is well within the skill of a person trained in the art
to create alternative DNA sequences encoding the same or
essentially the same, toxin. These variant DNA sequences are within
the scope of the subject invention. As used herein, reference to
"essentially the same" sequence refers to sequences, which have
amino acid substitutions, deletions, additions or insertions, which
do not materially affect pesticidal activity. Fragments retaining
pesticidal activity are also included in this definition.
[0119] A novel chimeric toxin of the subject invention has been
specifically exemplified herein. It should be readily apparent that
the subject invention comprises variants or equivalent toxins (and
nucleotide sequences encoding equivalent toxins) having the same or
similar pesticidal activity of the exemplified toxin. Equivalent
toxins will have amino acid homology with the exemplified toxin.
This amino acid homology will typically be greater than 75%,
preferably be greater than 90% and most preferably be greater than
95%. The amino acid homology will be highest in critical regions of
the toxin, which account for the biological activity or are
involved in the determination of three-dimensional configuration,
which ultimately is responsible for the biological activity. In
this regard, certain amino acid substitutions are acceptable and
can be expected if these substitutions are in regions, which are
not critical in biological activity or are conservative amino acid
substitutions, which do not affect the three-dimensional
configuration of the molecule. For example, amino acids may be
placed in the following classes: non-polar, uncharged polar, basic
and acidic. Conservative substitutions whereby an amino acid of one
class is replaced with another amino acid of the same class fall
within the scope of the subject invention so long as the
substitution does not materially alter the biological activity of
the .delta.-endotoxin. Table 3 as given here below provides a
listing of examples of amino acids belonging to each class.
TABLE-US-00004 TABLE 3 Class of Amino Acid Examples of Amino Acids
Non-polar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar
Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg,
His
[0120] In some instances, non-conservative substitutions can also
be made. The critical factor is that these substitutions must not
significantly detract from the biological activity of the toxin. It
is well within the skill of a person trained in the art of protein
engineering to substitute any amino acid of the chimeric toxin with
alanine. The substitution of any amino acid is safest, as alanine
is a typical amino acid. Such substitution is also well within the
scope of the invention.
[0121] A gene encoding the chimeric toxin of the subject invention
can be introduced into a wide variety of microbial or plant hosts.
Expression of the toxin gene results, directly or indirectly, in
the intracellular production and maintenance of the pesticidal
chimeric toxin. With suitable microbial hosts, e.g., Pseudomonas,
the microbes can be applied to the sites where the pests
proliferate. This will result into the control of the pest.
Alternatively, the microbe hosting the toxin gene can be treated
under conditions that prolong the activity of the toxin and
stabilize the cell. The treated cell, which retains the toxic
activity, then can be applied to the environment of the target
pest. Where the gene encoding the chimeric toxin is introduced via
a suitable vector into a microbial host, and said host is applied
to the environment in a living state, it is essential that certain
host microbes be used. Microorganism hosts are selected which are
known to occupy the "phytosphere" (phylloplane, phyllosphere,
rhizosphere, and/or rhizoplane) of one or more crops of interest.
These microorganisms are selected so as to be capable of
successfully competing in the particular environment (crop and
other insect habitats) with the wild-type microorganisms, provide
for stable maintenance and expression of the gene expressing the
polypeptide pesticide, and, desirably, provide for improved
protection of the pesticide from environmental degradation and
inactivation.
[0122] A large number of microorganisms are known to inhabit the
phylloplane (the surface of the plant leaves) and/or the
rhizosphere (the soil surrounding plant roots) of a wide variety of
important crops. These microorganisms include bacteria, algae, and
fungi. Of particular interest are microorganisms, such as bacteria,
e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella,
Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
Methylophilius, Agrobacterium, Acetobacter, Lactobacillus,
Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,
particularly yeast, e.g., genera Saccharomyces, Cryptococcus,
Kluyveromyces, Sporobolomyces, Rhodotorula and Aureobasidium. Of
the particular interest are such phytosphere bacterial species as
Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,
Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas
spheroids, Xanthomonas campestris, Rhozobium melioti, Alcaligenes
entrophus and Azotobacter vinlandii 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. A wide
variety of ways are available for introducing a gene encoding a
chimeric toxin into a microorganism host under conditions, which
allow for the stable maintenance and expression of the gene. These
methods are well known to those skilled in the art and are
described, for example, in U.S. Pat. No. 5,135,867, which is
incorporated herein by reference.
[0123] A plant transformation vector was constructed for the
development of transgenic plants. A plasmid pPK58 (having CaMV35S
promoter with duplicated enhancer) was digested with BamHI and
HindIII and pPK200 with HindIII and EcoRI to excise out CaMV35S
promoter with the duplicated enhancer and chimeric cry1E gene,
respectively. A triple ligation was carried for cloning of the two
fragments in pLITMUS38 cloning vector (New England Biolabs). The
plasmid, namely pPK59 had CaMV35S promoter with the duplicated
enhancer at the upstream of the chimeric gene. Restriction analysis
of pPK59 is shown in FIG. 2. The nos transcription terminator was
cloned at the downstream of the chimeric gene. DNA of nos
polyadenylation element was amplified using pBI101.1 as template
with suitable primers, which created MeI and EcoRI restriction
sites at the upstream and downstream, respectively. The plasmid
pPK59 was digested with EcoRI and the PCR product was cloned
following digestion with the MfeI and EcoRI restriction enzymes.
The clone in which EcoRI restriction site of the synthetic gene was
ligated to MfeI site of nos terminator (as they have compatible
ends), selected and named as pPK201. The correct orientation of nos
terminator was confirmed by restriction analysis and also by
sequencing. The expression cassette (the synthetic cry gene with
E-35S promoter and nos terminator) was cloned in Ti binary vector.
BamHI-EcoRI fragment of plasmid pPK201 was cloned in pBI101.1
replacing BamHI-EcoRI fragment (uidA gene and nos terminator) of
the plasmid. This binary vector was named as pPK202. The map of the
E. coli and plant expression vectors are shown in FIG. 3.
[0124] In order to study the efficacy of the chimeric Cry1E toxin
in plants, tobacco was selected for the expression. Agrobacterium
tumefaciens strain LBA 4404 containing the helper plasmid pAL4404
was transformed with the binary vector pPK202 following the
modified protocol of "electroporation of Agrobacterium" discussed
by Cangelosi et al. (1991) and transformed colony was selected on
antibiotics streptomycin, rifampicin and kanamycin. Agrobacterium
mediated transformation of Nicotiana tobacum cv. Patit Havana was
carried out following the method of Horsch et al., 1985 and the
transgenic plant were selected on the antibiotic kanamycin. The
presence of the gene encoding chimeric toxin was confirmed with PCR
and Southern analysis and the expression of the to transgene was
established with the RT-PCR, Western analysis and ELISA. ELISA
result displayed 0.5% expression of the toxin protein out of total
soluble leaf protein in a selected transgenic line. This high level
of the expression was the result of the designing of the gene in
which plant-preferred codons were exclusively used. Plant preferred
translation initiation context used in this study would also have
played an important role in achieving enhanced expression.
[0125] Insect bioassay was performed with two months old transgenic
plants and 1.sup.st and 5.sup.th instar larvae of Spodoptera
litura. (Please refer FIGS. 4-6). 15 cm.sup.2 leaf-discs of
transgenic and control plants were placed in cylindrical boxes
containing wet blotting paper at the bottom and ten 1.sup.st instar
larvae were released onto them. Mouths of the boxes were covered
with wet muslin cloth to maintain sufficient humidity and exchange
of air. The toxicity experiments were conducted in three replicates
at 25.+-.0.2.degree. C. and 16/8 h photoperiod was maintained. In
bioassay with 5.sup.th instar larvae, complete leaves of transgenic
and control plants were used. The leaf petiole was held in cotton
plug over a 250 ml flask containing 1/2 MS salt solution to
overcome wilting of the leaf. Five insect larvae were allowed to
feed on each leaf. The leaves of control plant were changed after
every 8 h and they were consumed completely by the insect
larvae.
[0126] The result (FIGS. 4 and 6) established that 1.sup.st as well
as 5.sup.th instar larvae died within 48-72 h. The amount of leaf
eaten by the 1.sup.st instar larvae was negligible as compare to
the control plant leaf discs, which were eaten voraciously.
Ingestion of approximately 1 cm.sup.2 of transgenic leaf was
sufficient to kill 5.sup.th instar larvae. These larvae appeared
moribund after 8-16 h of feeding on the transgenic leaf and finally
died within 2-3 days. Green coloured excreta with high water
content were noticed on leaf surface. Some of the larvae showed
heavy weight loss before death. In a separate experiment, different
instar larvae were allowed to feed on leaves of transgenic plant
for 1 h, 2 h, 4 h, 8 h and 16 h and then shifted to control plant
leaves. It was observed that 8 h of feeding on transgenic plants
was sufficient to cause 100% mortality of larvae in all stages of
development, even after feeding on non-transgenic plants. Ingestion
of very small amounts of the toxin by young larvae delayed their
pupation by 10-15 days from normal larval cycle of 15 days. The few
larvae that escaped mortality developed into flies. 40% of the
paired matings where such flies were used, gave eggs. However, the
eggs were sterile and failed to hatch. The total soluble protein
from transgenic tobacco plant was extracted and loaded on
SepharoseQ ion exchange column. The protein was eluted with
increasing gradient of sodium chloride and the peak containing
.delta.-endotoxin was pooled and desalted on G10 column. The eluted
protein was mixed in semi-synthetic diet. Similar protein
extraction and purification from the leaf of non-transgenic tobacco
plants was also performed and such plant protein served as control.
The toxicity experiment was conducted as discussed earlier.
EC.sub.50 for Spodoptera litura and Helicoverpa armigera was 37
ng/ml and 285 ng/ml of artificial diet. The result again confirmed
the efficacy of chimeric .delta.-endotoxin towards target insect
pests.
[0127] A novel .delta.-endotoxin for the control insect pests and a
gene for its high level expression in plants, which comprises
theoretical designing of a novel .delta.-endotoxin, herein named
chimeric Cry1E, strategically designed by replacing a polypeptide
domain (from position 530 to 587) of Cry1Ea protein by that of
Cry1Ca (from position 533 to 602), a novel 25 amino acid residues
long polypeptide at the C-terminus extreme of the protein for
stability, theoretical designing of the gene to express the
chimeric .delta.-endotoxin at a high level in plants, designing and
chemical synthesis of the oligonucleotides representing the
theoretically designed gene, assembly of oligonucleotides into
double stranded DNA, cloning and sequence analyses of the cloned
synthetic DNA, construction of vectors for the expression of
chimeric gene in E. coli and plants, expression of the synthetic
gene in E. coli, comparison of the toxicity of the chimeric protein
with the parental proteins against Spodoptera litura,
transformation of plant, for example tobacco with the chimeric
gene, high level expression of the engineered protein in transgenic
plants and evaluation of the potential of the chimeric toxin in
transgenic plants for protection against Spodoptera litura.
[0128] In still another embodiment of the present invention,
wherein amino acid no. 1 to 529 of Cry1Ea, 530 to 599 of Cry1Ca,
600 to 616 of Cry1Ea and 617 to 641 a novel polypeptide and its
structurally and/or functionally equivalent variants or fragments
thereof.
[0129] In still another embodiment of the present invention,
wherein the said toxin has an amino, acid sequence shown in SEQ ID
No.1; shows several fold higher toxicity to target lepidopterin
insects as compared to the parental toxins Cry1Ea and Cry1Ca.
[0130] In still another embodiment of the present invention,
wherein chimeric cry1E gene which encodes a chimeric toxin having
activity against lepidopteran insects, has nucleotide sequence
shown in SEQ ID No 2 or its fragment thereof.
[0131] In still another embodiment of the present invention,
wherein a process of controlling lepidopteran pests employing the
protein, with an effective amount of the toxin used as such or as a
component of a chemical or microbial formulation.
[0132] In still another embodiment of the present invention,
wherein a recombinant DNA transfer vector comprising a
polynucleotide sequence, which encodes a toxin having activity
against lepidopteran insects, wherein said polynucleotide sequence
has the nucleotide sequence of SEQ ID NO. 2 or fragments
thereof.
[0133] In still another embodiment of the present invention,
wherein a recombinant host transformed with the gene.
[0134] In still another embodiment of the present invention,
wherein the said host is a microbe for example Escherichia coli,
Pseudomonas, Yeast, Cyanobacteria and/or other microbes.
[0135] In still another embodiment of the present invention,
wherein said transformed host is a plant for example tobacco,
cotton, chickpea, pegeonpea, groundnut, cauliflower, cabbage,
chilli, capsicum and/or other plants, which expresses the toxin,
wherein said toxin has the amino acid sequence of SEQ ID NO. 1, or
its variants with equivalent activity against lepidopteran
insects.
[0136] In further embodiment of the present invention, instant
Application clearly states that in proteins, more particularly in
the field of endotoxins, the high homology of the sequence is not
found to make any significant difference in activity. The
above-referred work on of endotoxins Cry1Aa1 to Cry1Aa6 clearly
reflect the essence of this work. In the instant Application, the
applicant has observed extraordinarily high insecticidal activity.
Further, the homology of 70% and above in the sequence of chimeric
protein Cry1E of the instant Application is also found to show no
significant change in the activity. This means that the proteins
with sequence homology of 70% and above for chimeric protein Cry 1E
are used as insecticidal agents.
[0137] The following examples are given by way of illustrations and
therefore should not be construed to limit the scope of the present
invention.
Example 1
Comparative Toxicity of the Novel Chimeric Cry1E Expressed in E.
coli to Larvae of Lepidopteran Insect Pests
[0138] A chimeric .delta.-endotoxin, 616 amino acid residues long,
herein said chimeric Cry1E was strategically designed by replacing
a polypeptide domain (from position 530 to 587) of Cry1Ea protein
by that of Cry1Ca (from position 533 to 602). A polypeptide of 25
amino acid residues was additionally included at the C-terminus
extreme as described earlier. A 1.99 kb nucleotide sequence was
theoretically designed to code for the above-mentioned chimeric
.delta.-endotoxin. The codons for each amino acid were distributed
evenly to avoid temporary deficiency of the tRNA during
translation. Several 6-base cutter restriction enzyme sites were
created in the designed gene. BamHI, HindIII and NcoI restriction
sites were created at 5'-end and EcoRI at the 3'-end of the
designed gene. The gene was divided into 58 overlapping
oligonucleotides (40 to 65 nucleotides long). Each oligonucleotide
had 13-18 nucleotide long overlap with the immediately adjacent
oligonucleotides on the complementary strand (T.sub.m between
48-50.degree. C.). Oligonucleotides were synthesised on a DNA
synthesiser (Gene Assembler Special, Pharmacia, Sweden) at 200
nmole scale and purified on denaturing urea-PAGE. All 58
oligonucleotides were assembled into the double-stranded DNA,
herein said chimeric cry1E gene following the ligation-free gene
synthesis method of Singh et al. (1996) and as shown in FIG. 1. The
DNA was digested with HindIII and EcoRI restriction enzyme and
cloned in pBluescriptII SK(+) (Stratagene). The plasmid was named
as pPK200. The nucleotide sequence of the synthetic DNA was
confirmed by sequencing of the cloned synthetic DNA on automated
DNA sequencing system (Applied Biosystems model 373).
[0139] A cassette was constructed for the expression of the
chimeric toxin in E. coli under control of T7 promoter. For this,
plasmid pPK200 was digested with the restriction enzymes NcoI and
BanHI and cloned in expression vector pET-19b (Novagen). The
plasmid was named as pPK206. DNA encoding toxin portion of Cry1Ea
and Cry1Ca were amplified with polymerase chain reaction, using
suitable primers, which created NcoI and BamHI restriction sites at
the upstream and the downstream of the amplicon, respectively in
both the DNA. The amplified products were cloned at NcoI and BamHI
site in the same vector (pET-19b). The constructs having Cry1Ea
toxin DNA was named as pPK141 and Cry1Ca as pPK135 as described
earlier. BL21DE3 strain of E. coli was transformed with the
constructs pPK141, pPK135 and pPK206. The toxin proteins were
expressed by induction with appropriate concentrations of IPTG. The
expression was carried out at 15.degree. C. to avoid any possible
mis-folding of the toxins. The toxin proteins were quantified
densitometrically on the denaturing polyacrylamide gel. Serial
dilutions of the toxins were mixed in semi-synthetic diet. Total E.
coli protein served as control in the diet. Fifteen neonatal larvae
of Spodoptera litura were released onto the cakes of the diet
mixture in a 100-ml beaker and the mouth of the beaker was covered
with muslin cloth to allow gas exchange. Each experiment was
conducted in 6 replicates. The diet was changed after every
alternate day. Bio-assay was conducted with 16/8 h photoperiod at
25.+-.0.2.degree. C. Toxicity data was recorded after 7 days of the
feeding. EC.sub.50 was determined by standard log-probit analysis.
All three proteins were tested simultaneously. The representative
results are presented in Table 4 as given here below.
TABLE-US-00005 TABLE 4 .delta.-endotoxins(S) EC.sub.50 (.mu.g/ml
semi-synthetic diet) Cry1Ea >108 Cry1Ca 29.48 .+-. 1.77 Chimeric
Cry1E 6.27 .+-. 0.59
[0140] The result showed that the chimeric toxin was several fold
more toxic over Cry1Ea and more than four fold toxic over Cry1Ca
protein. Cry1Ea toxin protein failed to cause any mortality or
growth retardation of the Spodoptera larvae. The result established
the successful engineering of the Cry1Ea toxin for converting it
into a biologically active improved toxin. The engineered protein
was more toxic than Cry1Ca protein, which is the best-known
.delta.-endotoxoin against Spodoptera sp.
[0141] A similar toxicity experiment was conducted with the larvae
of Helicoverpa armigera. In this case, 72 h old larvae were
released on semi-synthetic diet containing one of the three
proteins and only one larvae was released in each box. Weight loss
was recorded after 7 days of feeding. The representative results
are presented in Table 5 as shown here below.
TABLE-US-00006 TABLE 5 .delta.-endotoxins(S) EC.sub.50 (.mu.g/ml
semisynthetic diet) Cry1Ea >176 Cry1Ca 136.22 .+-. 8.77 Chimeric
Cry1E 26.71 .+-. 1.39
[0142] The result shows that the novel chimeric 8-endotoxoin
designed by us is not only more toxic to Spodoptera but also
effective against Helicoverpa. The designing has widened the host
range of the toxin as well as substantially improved toxicity over
the parental proteins.
Example 2
High Larval Toxicity of the Transgenic Plants Expressing Novel
Chimeric Cry1E Protein
[0143] In order to establish efficacy of the novel chimeric toxin
in plants, a, plant transformation vector was constructed for the
development of transgenic plants. A plasmid pPK58 (having CaMV35S
promoter with duplicated enhancer) was digested with BamHI and
HindIII and pPK200 with HindIII and EcoRI to excise out CaMV35S
promoter with the duplicated enhancer and chimeric cry1E gene,
respectively. A triple ligation was carried for the cloning of the
two fragments in pLITMUS38 cloning vector (New England Biolabs).
The plasmid was named pPK59, which had CaMV35S promoter with the
duplicated enhancer at the upstream of the chimeric gene. The nos
transcription terminator was cloned at the downstream of the
chimeric gene nos polyadenylation element was amplified using
pBI101.1 as template with suitable primers, which created MfeI and
EcoRI restriction sites at the upstream and downstream,
respectively. The plasmid pPK59 was digested with EcoRI and the PCR
product was cloned following the digestion with the MfeI and EcoRI
restriction enzymes. The clone in which EcoRI restriction site of
the synthetic gene ligated to MfeI site of nos terminator (as they
have compatible ends) was selected and named as pPK201. The correct
orientation of nos terminator was confirmed by restriction analysis
and also by DNA sequencing. The expression cassette (the synthetic
cry gene with E-35S promoter and nos terminator) was cloned in Ti
binary vector. BamHI-EcoRI fragment of plasmid pPK201 was cloned in
pBI101.1 replacing BamHI-EcoRI fragment (uidA gene and nos
terminator) of the plasmid. This binary vector was named as pPK202.
The construction of E. coli and plant expression vector is
schematically presented in FIG. 3. The construct had polynucleotide
sequences TAAACCATG GCT (SEQ ID NO:9) as plant preferred
translation initiation context, TAA TGA were introduced in
synthetic gene for translational termination. Agrobacterium
tumefaciens strain LISA 4404 containing helper plasmid pAL4404 was
transformed with binary vector pPK202 following the modified
protocol of "electroporation of Agrobacterium" discussed by
Cangelosi et al. (1991) and transformed colony was selected on
antibiotics streptomycin, rifampicin and kanamycin. Agrobacterium
mediated transformation of Nicotiana tabacum cv. Patit Havana was
carried out following the method of Horsch et al., 1985 and the
transgenic plant were selected on the antibiotic kanamycin. The
presence of the gene encoding chimeric toxin was confirmed with the
PCR and Southern Analysis and the expression of the transgene was
established with the RT-PCR, Western analysis and ELISA. ELISA
result established 0.5% expression of the toxin protein in total
soluble leaf protein in the transgenic line selected for these
experiments. This high level of the expression was the result of
the designing of the gene in which plant-preferred codons were
exclusively used. Plant preferred translation initiation context
also would have played an important role in the expression. Insect
bioassay was performed with the leaves of two-month-old transgenic
plants and neonatal larvae of Spodoptera litura. 15 cm.sup.2
leaf-discs of transgenic and control plants were placed in
cylindrical boxes containing wet blotting paper at the bottom and
ten 1.sup.st instar larvae were released onto them. Mouths of the
boxes were covered with wet muslin cloth to maintain sufficient
humidity and to allow the exchange of air. The toxicity experiments
were conducted in six replicates at 25.+-.0.2.degree. C. and 16/8 h
photoperiod was maintained. The result showed (FIG. 4) that the
transgenic plants expressing novel chimeric protein were highly
toxic to the neonatal larvae of Spodoptera litura and cause 100%
mortality within 48 h of feeding. The damage of leaf-discs by the
insect larvae was negligible as compared to control plant
leaf-discs, which were almost completely eaten away. High level of
the protection of the transgenic plant and mortality of Spodoptera
larvae upon feeding on transgenic plants again established the
efficacy of the chimeric toxin and the transgenic plants.
Example 3
High Toxicity of the Chimeric Cry1E Protein to Larvae of Spodoptera
sp. in all the Stages of their Development
[0144] A bioassay was conducted on 1.sup.st (3 days old), 3.sup.rd
(7 days old) and 5.sup.th (12 days old) instar larvae to
established the efficacy of the engineered protein expressed in the
transgenic plants. Bioassay with 1.sup.st instar larvae has been
discussed in example 2. Complete leaves of transgenic and control
plants were used for feeding the advanced stage larvae. The leaf
petiole was held in cotton plug over a 250 ml flask containing 1/2
MS salt solution to overcome wilting of the leaf. 5 insect larvae
were allowed to feed on each leaf. The leaves of control plant fed
by 3.sup.rd (FIG. 5) and 5.sup.th (FIG. 6) instar larvae were
changed after 16 h and 8 h, respectively, as they were consumed
completely by the insect larvae. The result established that
feeding on transgenic leaf causes mortality of larvae in all the
developmental stages within 48 h. Ingestion of approximately 1 cm'
of transgenic leaf was sufficient to kill 5.sup.th instar larvae.
These larvae appeared moribund after 8-16 h of feeding on the
transgenic leaf and finally died within 2 days. Green coloured
excreta with high water content were noticed on leaf surface. Some
of the larvae showed heavy weight loss before death.
[0145] In a separate experiment, different instar larvae were fed
on leaves of transgenic plants for 1 h, 2 h, 4 h, 8 h and 16 h and
then shifted to control plant leaves. It was observed that 4 h of
feeding on transgenic plants was sufficient to cause 100% mortality
of larvae in all stages of development, even when they were
subsequently fed on non-transgenic plants. Ingestion of very small
amounts of the toxin by young larvae delayed their pupation by
10-15 days beyond the normal larval cycle of 15 days. The few
larvae that escaped mortality developed into flies. 40% of the
paired matings using such flies gave eggs. However, the eggs were
sterile and failed to hatch. The toxicity of .delta.-endotoxin to
advance stage insect larvae has not been reported in literature
till date. The high level of toxicity may be due to higher
stability of chimeric toxin in the mid gut of insect larvae or
improved receptor binding and pore-forming ability of
.delta.-endotoxin. The example again established the potential of
the chimeric toxin against Spodoptera sp. Since Helicoverpa does
not prefer to cat tobacco leaf, toxicity experiment with transgenic
tobacco plants could not be conducted on Helicoverpa.
Example 4
High Toxicity of the Novel Chimeric Cry1E Prepared Front Leaves of
Transgenic Tobacco Plants Expressing .delta.-Endotoxin
[0146] Total soluble protein was prepared from leaves of transgenic
tobacco. Fresh leaf tissue was powered under liquid N2 and then
suspended in 5 volumes of protein extraction buffer (TrisCl, 20 mM,
pH 9.5; EDTA 2 mM, pH 8.0; NaCl, 50 mM; DTT, 1 mM; PVP 2% and PMSF,
100 mM). The suspension was mixed well and centrifuged twice
(20,000.times.g, 20 min and 4.degree. C.). The supernatant was
loaded on Sepharose Q column (10 cm.times.2.5 cm). The protein was
eluted with increasing gradient of NaCl in extraction buffer and
100 fractions of 5 ml were collected. The .delta.-endotoxin was
detected with ELISA. The fractions containing the .delta.-endotoxin
were pooled. A known amount of the plant-purified .delta.-endotoxin
was mixed in semi-synthetic diet. The toxicity trials were
conducted with 3-day old larvae of Spodoptera litura and
Helicoverpa armigera, as described in previous examples. The result
showed that EC.sub.50 (the concentration required for 50% killing)
for Spodoptera and Helicoverpa were 42.39.+-.1.72 ng/ml and
283.11.+-.8.29 ng/ml of semi-synthetic diet. The result further
established the high level of toxicity to the larvae of Spodoptera
and Helicoverpa. The point is noteworthy that insecticidal crystal
protein made in plant tissue is much more toxic as compared to the
same protein made in E. coli. Probably the .delta.-endotoxin folds
much better in plant cytoplasm, the role of some unidentified
chaperons in such folding cannot be overruled.
Example 5
Stability of Chimeric .delta.-Endotoxin in Plant Tissue
[0147] Total soluble leaf protein of transgenic plant was extracted
as discussed earlier and incubated at 4.degree. C. and 28.degree.
C. They were used for toxicity trials. The samples were taken out
after every two days in case of former and every day in case of
later. The total crude protein was mixed in semi synthetic diet and
toxicity experiment was carried out with neonatal larvae of
Spodoptera litura. The insect mortality data was recorded after 7
days of feeding and LC.sub.50 was calculated. The results are shown
in Tables 6, and 7 here below.
TABLE-US-00007 TABLE 6 Incubation period of crude protein LD.sub.50
(in ng/ml of semi- S. No. at 4.degree. C. (in days) synthetic diet)
1. 2 48.71 .+-. 2.4 2. 4 57.87 .+-. 2.96 3. 6 66.44 .+-. 3.65 4. 8
70.19 .+-. 3.55 5. 12 73.82 .+-. 3.1 6. 16 74.37 .+-. 3.67
TABLE-US-00008 TABLE 7 Incubation period of crude protein LD.sub.50
(in ng/ml of semi- S. No. at 28.degree. C. (in days) synthetic
diet) 1. 1 76.44 .+-. 3.3 2. 2 98.13 .+-. 4.6 3. 3 143.46 .+-. 8.5
4. 4 -- 5. 5 --
[0148] The result established the stability of chimeric
.delta.-endotoxin designed by us against the plant proteases. The
chimeric toxin was stable for more than 16 days at 4.degree. C. and
3 days at 28.degree. C., and caused more than 80% mortality.
Increase in LC.sub.50 with time was presumably due to some
degradation of the toxin.
[0149] Main advantages of the present invention are: [0150] 1.
Cry1Ea .delta.-endotoxin was engineered to obtain a novel chimeric
toxin, herein said chimeric Cry1E. The toxicity of the chimeric
protein was several fold higher as compared to the parent toxins or
other .delta.-endotoxin reported to function against Spodoptera.
Its larvicidal activity was very high when it is made in plants.
[0151] 2. The gene encoding chimeric toxin was designed to express
both in E. coli and plants. Hence, it can be used in the
engineering of a microbe for the expression of chimeric toxin,
which can be used in the preparation of the microbial formulation.
The same gene can also be used in the genetic engineering of the
plants for the trade of insect resistance. We have shown that the
sequence designed by us gives very high level of expression (0.5%
of the total soluble protein) of the chimeric toxin in transgenic
tobacco and cotton leaves (results not included). [0152] 3. The
transgenic plants expressing the chimeric toxin exhibited very high
degree of protection against the larvae of Spodoptera litura in all
developmental stages. They died within 2-3 days of feeding on the
transgenic plants. Such high level of toxicity of transgenic plants
against any lepidopteran insect has not been reported till date.
This protein may also be effective against many other insect
larvae. Our results show that the toxin was effective against
Helicoverpa also. [0153] 4. Potential of the chimeric toxin in
transgenic plants was further established by short-term feeding on
transgenic plants. Feeding for 4 hours caused 100% mortality of
Spodoptera larvae at all the developmental stages. The feeding for
extremely short (up to one hour) periods, delayed larval
development and interfered with metamorphosis. Such protein may be
extremely valuable in protecting agronomically important crops and
forests. The gene coding chimeric toxin can be used in the
development of transgenic plants and/or for production of the toxin
in a microbe, which can be used in microbial formulations. [0154]
5. Since the transgenic plants expressing the novel chimeric toxin
caused 100% mortality of Spodoptera larvae within a very short
period of feeding, the probability of the development of resistance
in insects against this .delta.-endotoxin will be extremely low.
Sequence CWU 1
1
101641PRTBacillus thuringiensis 1Met Ala Ile Val Asn Asn Gln Asn
Gln Cys Val Pro Tyr Asn Cys Leu 1 5 10 15Asn Asn Pro Glu Asn Glu
Ile Leu Asp Ile Glu Arg Ser Asn Ser Thr 20 25 30Val Ala Thr Asn Ile
Ala Leu Glu Ile Ser Arg Leu Leu Ala Ser Ala 35 40 45Thr Pro Ile Gly
Gly Ile Leu Leu Gly Leu Phe Asp Ala Ile Trp Gly 50 55 60Ser Ile Gly
Pro Ser Gln Trp Asp Leu Phe Leu Glu Gln Ile Glu Leu65 70 75 80Leu
Ile Asp Gln Lys Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser 85 90
95Arg Leu Glu Gly Ile Ser Ser Leu Tyr Gly Ile Tyr Thr Glu Ala Phe
100 105 110Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Lys Glu
Glu Met 115 120 125Arg Thr Gln Phe Asn Asp Met Asn Ser Ile Leu Val
Thr Ala Ile Pro 130 135 140Leu Phe Ser Val Gln Asn Tyr Gln Val Pro
Phe Leu Ser Val Tyr Val145 150 155 160Gln Ala Ala Asn Leu His Leu
Ser Val Leu Arg Asp Val Ser Val Phe 165 170 175Gly Gln Ala Trp Gly
Phe Asp Ile Ala Thr Ile Asn Ser Arg Tyr Asn 180 185 190Asp Leu Thr
Arg Leu Ile Pro Ile Tyr Thr Asp Tyr Ala Val Arg Trp 195 200 205Tyr
Asn Thr Gly Leu Asp Arg Leu Pro Arg Thr Gly Gly Leu Arg Asn 210 215
220Trp Ala Arg Phe Asn Gln Phe Arg Arg Glu Leu Thr Ile Ser Val
Leu225 230 235 240Asp Ile Ile Ser Phe Phe Arg Asn Tyr Asp Ser Arg
Leu Tyr Pro Ile 245 250 255Pro Thr Ser Ser Gln Leu Thr Arg Glu Val
Tyr Thr Asp Pro Val Ile 260 265 270Asn Ile Thr Asp Tyr Arg Val Gly
Pro Ser Phe Glu Asn Ile Glu Asn 275 280 285Ser Ala Ile Arg Ser Pro
His Leu Met Asp Phe Leu Asn Asn Leu Thr 290 295 300Ile Asp Thr Asp
Leu Ile Arg Gly Val His Tyr Trp Ala Gly His Arg305 310 315 320Val
Thr Ser His Phe Thr Gly Ser Ser Gln Val Ile Thr Thr Pro Gln 325 330
335Tyr Gly Ile Thr Ala Asn Ala Glu Pro Arg Arg Thr Ile Ala Pro Ser
340 345 350Thr Phe Pro Gly Leu Asn Leu Phe Tyr Arg Thr Leu Ser Asn
Pro Phe 355 360 365Phe Arg Arg Ser Glu Asn Ile Thr Pro Thr Leu Gly
Ile Asn Val Val 370 375 380Gln Gly Val Gly Phe Ile Gln Pro Asn Asn
Ala Glu Val Leu Tyr Arg385 390 395 400Ser Arg Gly Thr Val Asp Ser
Leu Asn Glu Leu Pro Ile Asp Gly Glu 405 410 415Asn Ser Leu Val Gly
Tyr Ser His Arg Leu Ser His Val Thr Leu Thr 420 425 430Arg Ser Leu
Tyr Asn Thr Asn Ile Thr Ser Leu Pro Thr Phe Val Trp 435 440 445Thr
His His Ser Ala Thr Asn Thr Asn Thr Ile Asn Pro Asp Ile Ile 450 455
460Thr Gln Ile Pro Leu Val Lys Gly Phe Arg Leu Gly Gly Gly Thr
Ser465 470 475 480Val Ile Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile
Leu Arg Arg Asn 485 490 495Thr Ile Gly Glu Phe Val Ser Leu Gln Val
Asn Ile Asn Ser Pro Ile 500 505 510Thr Gln Arg Tyr Arg Leu Arg Phe
Arg Tyr Ala Ser Ser Arg Asp Ala 515 520 525Arg Val Ile Val Leu Thr
Gly Ala Ala Ser Thr Gly Val Gly Gly Gln 530 535 540Val Ser Val Asn
Met Pro Leu Gln Lys Thr Met Glu Ile Gly Glu Asn545 550 555 560Leu
Thr Ser Arg Thr Phe Arg Tyr Thr Asp Phe Ser Asn Pro Phe Ser 565 570
575Phe Arg Ala Asn Pro Asp Ile Ile Gly Ile Ser Glu Gln Pro Leu Phe
580 585 590Gly Ala Gly Ser Ile Ser Ser Gly Glu Leu Tyr Ile Asp Lys
Ile Glu 595 600 605Leu Ile Leu Ala Asp Ala Thr Phe Lys Arg Arg Arg
Trp Ser Val His 610 615 620Lys Ala Ser Arg Pro Leu His Leu His Gln
Gln Ala Gly Leu Ala Ala625 630 635 640Asp21990DNABacillus
thuringiensis 2cccgcatgcc ccgggggatc caagctttaa accatggcta
tcgttaacaa ccagaaccag 60tgcgtccctt acaattgcct caacaaccca gagaacgaga
tcttggacat cgaaagatcc 120aattctaccg tggccaccaa cattgctctt
gagatttcca gattgctcgc tagcgcaact 180cccattggtg gcatcctcct
tggattgttc gacgccattt ggggttccat cggaccatca 240caatgggatc
tcttccttga acagatcgag ttgctcattg accagaagat cgaagagttt
300gctaggaacc aggcaattag ccgtctcgag gggatctctt ccctttacgg
aatctataca 360gaggccttca gagagtggga agctgaccct actaatccag
cattgaagga agagatgcgt 420actcaattca acgatatgaa ctctatcttg
gtcaccgcca ttcctctctt ctcagtgcag 480aactaccaag tgccattcct
ctccgtctat gttcaagctg caaacttgca cctttctgtc 540cttcgcgacg
tgtccgtctt tggtcaagcc tggggcttcg atatcgctac tatcaactcc
600cgttacaacg acctcacaag gttgattcct atctacactg actacgctgt
tagatggtac 660aatactgggc ttgacagact cccacgtacc ggcggattga
ggaattgggc tcgcttcaac 720cagtttaggc gtgagctcac cattagcgtg
ttggacatca tttccttctt cagaaactac 780gactctagac tttatcctat
tccaactagt tctcaactca ccagggaggt ctacaccgat 840cctgtgatca
acattaccga ctatcgtgtg ggtccctcct tcgagaacat tgaaaacagc
900gctatcagat ctccacacct tatggacttc ctcaataact tgactatcga
tacagacctt 960atcagaggtg ttcactactg ggctggccat agggtcacct
ctcactttac cggtagttcc 1020caagtgatca caacccctca atacggaatt
actgccaacg cagagccaag acgtaccatt 1080gctccaagta cctttcccgg
gttgaacctc ttctaccgca cattgtcaaa tccattcttc 1140aggagatctg
agaacatcac ccctaccctt gggatcaacg ttgtccaggg agtgggtttc
1200atccagccaa acaatgctga ggtgctctac aggtctagag gcacagtgga
ctccttgaac 1260gaacttccaa ttgacggtga gaactcactc gtcggataca
gtcaccgtct tagccacgtt 1320actttgacca ggtctctcta taacactaat
atcactagtt tgcccacctt cgtgtggact 1380caccactcag ccaccaacac
aaacactatc aatcccgata tcattacaca aatccccctt 1440gtcaagggct
tccgcttggg tggagggacc tccgtcatta aagggcccgg attcaccggt
1500ggcgatatcc tccgtagaaa caccattggt gagtttgtgt ccctccaggt
taacattaac 1560tctcctatca cacaaaggta ccgtcttagg ttccgctacg
cttcctctag agacgcaaga 1620gtcattgtgc ttaccggtgc cgcttccaca
ggagtcggtg gccaagtcag cgttaacatg 1680ccattgcaaa agactatgga
gatcggagag aacctcacta gtagaacctt caggtatacc 1740gacttctcta
accctttctc cttccgtgct aacccagata tcattggcat cagcgaacaa
1800cctctcttcg gcgccggctc catcagctct ggtgaactct acatcgataa
gatcgagttg 1860atccttgctg acgccacatt caagaggaga cgatggagcg
tgcacaaagc ctcacgccct 1920cttcacctcc accaacaagc tggactcgct
gctgattaat gagaattcgg atccaagctt 1980gggcccgctc 19903609PRTBacillus
thuringiensis 3Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr
Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu
Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu
Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val
Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln
Trp Asp Ala Phe Pro Val Gln Ile65 70 75 80Glu Gln Leu Ile Asn Gln
Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95Ile Ser Arg Leu Glu
Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110Ser Phe Arg
Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu
Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135
140Ile Pro Leu Leu Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser
Val145 150 155 160Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
Arg Asp Val Ser 165 170 175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala
Ala Thr Ile Asn Ser Arg 180 185 190Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205Arg Trp Tyr Asn Thr Gly
Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220Asp Trp Val Arg
Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val225 230 235 240Leu
Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250
255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Met Ala Gln Arg
Ile Glu 275 280 285Gln Asn Ile Arg Gln Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr 290 295 300Ile Tyr Thr Asp Val His Arg Gly Phe Asn
Tyr Trp Ser Gly His Gln305 310 315 320Ile Thr Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe Ala Phe Pro 325 330 335Leu Phe Gly Asn Ala
Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu 340 345 350Thr Gly Leu
Gly Ile Phe Arg Thr Leu Ser Ser Pro Leu Tyr Arg Arg 355 360 365Ile
Ile Leu Gly Ser Gly Pro Asn Asn Gln Glu Leu Phe Val Leu Asp 370 375
380Gly Thr Glu Phe Ser Phe Ala Ser Leu Thr Thr Asn Leu Pro Ser
Thr385 390 395 400Ile Tyr Arg Gln Arg Gly Thr Val Asp Ser Leu Asp
Val Ile Pro Pro 405 410 415Gln Asp Asn Ser Val Pro Pro Arg Ala Gly
Phe Ser His Arg Leu Ser 420 425 430His Val Thr Met Leu Ser Gln Ala
Ala Gly Ala Val Tyr Thr Leu Arg 435 440 445Ala Pro Thr Phe Ser Trp
Gln His Arg Ser Ala Glu Phe Asn Asn Ile 450 455 460Ile Pro Ser Ser
Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr Asn465 470 475 480Leu
Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly 485 490
495Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val
500 505 510Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile
Arg Tyr 515 520 525Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile
Asp Gly Arg Pro 530 535 540Ile Asn Gln Gly Asn Phe Ser Ala Thr Met
Ser Ser Gly Ser Asn Leu545 550 555 560Gln Ser Gly Ser Phe Arg Thr
Val Gly Phe Thr Thr Pro Phe Asn Phe 565 570 575Ser Asn Gly Ser Ser
Val Phe Thr Leu Ser Ala His Val Phe Asn Ser 580 585 590Gly Asn Glu
Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val 595 600
605Thr4609PRTBacillus thuringiensis 4Met Asp Asn Asn Pro Asn Ile
Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val
Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile
Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val
Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly
Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Pro Val Gln Ile65 70 75
80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala
Glu 100 105 110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala
Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser
Ala Leu Thr Thr Ala 130 135 140Ile Pro Leu Leu Ala Val Gln Asn Tyr
Gln Val Pro Leu Leu Ser Val145 150 155 160Tyr Val Gln Ala Ala Asn
Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190Tyr Asn
Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200
205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu
Thr Val225 230 235 240Leu Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp
Ser Arg Arg Tyr Pro 245 250 255Ile Arg Thr Val Ser Gln Leu Thr Arg
Glu Ile Tyr Thr Asn Pro Val 260 265 270Leu Glu Asn Phe Asp Gly Ser
Phe Arg Gly Met Ala Gln Arg Ile Glu 275 280 285Gln Asn Ile Arg Gln
Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300Ile Tyr Thr
Asp Val His Arg Gly Phe Asn Tyr Trp Ser Gly His Gln305 310 315
320Ile Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Ala Phe Pro
325 330 335Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val Leu Val
Ser Leu 340 345 350Thr Gly Leu Gly Ile Phe Arg Thr Leu Ser Ser Pro
Leu Tyr Arg Arg 355 360 365Ile Ile Leu Gly Ser Gly Pro Asn Asn Gln
Glu Leu Phe Val Leu Asp 370 375 380Gly Thr Glu Phe Ser Phe Ala Ser
Leu Thr Thr Asn Leu Pro Ser Thr385 390 395 400Ile Tyr Arg Gln Arg
Gly Thr Val Asp Ser Leu Asp Val Ile Pro Pro 405 410 415Gln Asp Asn
Ser Val Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser 420 425 430His
Val Thr Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr Leu Arg 435 440
445Ala Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu Phe Asn Asn Ile
450 455 460Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser
Thr Asn465 470 475 480Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro
Gly Phe Thr Gly Gly 485 490 495Asp Ile Leu Arg Arg Thr Ser Pro Gly
Gln Ile Ser Thr Leu Arg Val 500 505 510Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr Arg Val Arg Ile Arg Tyr 515 520 525Ala Ser Thr Thr Asn
Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro 530 535 540Ile Asn Gln
Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu545 550 555
560Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe
565 570 575Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe
Asn Ser 580 585 590Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val
Pro Ala Glu Val 595 600 605Thr5609PRTBacillus thuringiensis 5Met
Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10
15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu
Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp
Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu
Val Gln Ile65 70 75 80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe
Ala Arg Asn Gln Ala 85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu
Tyr Gln Ile Tyr Ala Glu 100 105 110Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe
Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140Ile Pro Leu Leu
Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val145 150 155 160Tyr
Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170
175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg 210 215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg
Glu Leu Thr Leu Thr Val225 230 235 240Leu Asp Ile Val Ala Leu Phe
Ser Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu
Ile Tyr Thr Asn Pro Val 260 265 270Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly Met Ala Gln Arg Ile Glu 275 280 285Gln Asn Ile Arg Gln Pro
His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300Ile Tyr Thr Asp
Val His Arg Gly Phe Asn Tyr Trp Ser Gly His Gln305 310 315 320Ile
Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Ala Phe Pro 325 330
335Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu
340 345 350Thr Gly Leu Gly Ile Phe Arg Thr Leu Ser Ser Pro Leu Tyr
Arg Arg 355 360 365Ile Ile Leu Gly Ser Gly Pro Asn Asn Gln Glu Leu
Phe Val Leu Asp 370 375 380Gly Thr Glu Phe Ser Phe Ala Ser Leu Thr
Thr Asn Leu Pro Ser Thr385 390 395 400Ile Tyr Arg Gln Arg Gly Thr
Val Asp Ser Leu Asp Val Ile Pro Pro 405 410 415Gln Asp Asn Ser Val
Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser 420 425 430His Val Thr
Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr Leu Arg 435 440 445Ala
Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu Phe Asn Asn Ile 450 455
460Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr
Asn465 470 475 480Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly
Phe Thr Gly Gly 485 490 495Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln
Ile Ser Thr Leu Arg Val 500 505 510Asn Ile Thr Ala Pro Leu Ser Gln
Arg Tyr Arg Val Arg Ile Arg Tyr 515 520 525Ala Ser Thr Thr Asn Leu
Gln Phe His Thr Ser Ile Asp Gly Arg Pro 530 535 540Ile Asn Gln Gly
Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu545 550 555 560Gln
Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe 565 570
575Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser
580 585 590Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala
Glu Val 595 600 605Thr6609PRTBacillus thuringiensis 6Met Asp Asn
Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser
Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25
30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile
Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val
Gln Ile65 70 75 80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala
Arg Asn Gln Ala 85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr
Gln Ile Tyr Ala Glu 100 105 110Ser Phe Arg Glu Trp Glu Ala Asp Pro
Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn
Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140Ile Pro Leu Leu Ala
Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val145 150 155 160Tyr Val
Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170
175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr
Ala Val 195 200 205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg 210 215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg
Glu Leu Thr Leu Thr Val225 230 235 240Leu Asp Ile Val Ala Leu Phe
Ser Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255Ile Arg Thr Val Ser
Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270Leu Glu Asn
Phe Asp Gly Ser Phe Arg Gly Met Ala Gln Arg Ile Glu 275 280 285Gln
Asn Ile Arg Gln Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295
300Ile Tyr Thr Asp Val His Arg Gly Phe Asn Tyr Trp Ser Gly His
Gln305 310 315 320Ile Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu
Phe Ala Phe Pro 325 330 335Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro
Pro Val Leu Val Ser Leu 340 345 350Thr Gly Leu Gly Ile Phe Arg Thr
Leu Ser Ser Pro Leu Tyr Arg Arg 355 360 365Ile Ile Leu Gly Ser Gly
Pro Asn Asn Gln Glu Leu Phe Val Leu Asp 370 375 380Gly Thr Glu Phe
Ser Phe Ala Ser Leu Thr Thr Asn Leu Pro Ser Thr385 390 395 400Ile
Tyr Arg Gln Arg Gly Thr Val Asp Ser Leu Asp Val Ile Pro Pro 405 410
415Gln Asp Asn Ser Val Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser
420 425 430His Val Thr Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr
Leu Arg 435 440 445Ala Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu
Phe Asn Asn Ile 450 455 460Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro
Leu Thr Lys Ser Thr Asn465 470 475 480Leu Gly Ser Gly Thr Ser Val
Val Lys Gly Pro Gly Phe Thr Gly Gly 485 490 495Asp Ile Leu Arg Arg
Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val 500 505 510Asn Ile Thr
Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr 515 520 525Ala
Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro 530 535
540Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn
Leu545 550 555 560Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr
Pro Phe Asn Phe 565 570 575Ser Asn Gly Ser Ser Val Phe Thr Leu Ser
Ala His Val Phe Asn Ser 580 585 590Gly Asn Glu Val Tyr Ile Asp Arg
Ile Glu Phe Val Pro Ala Glu Val 595 600 605Thr7609PRTBacillus
thuringiensis 7Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr
Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu
Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu
Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val Pro Gly Ala Gly Phe Val
Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly Ile Phe Gly Pro Ser Gln
Trp Asp Ala Phe Leu Val Gln Ile65 70 75 80Glu Gln Leu Ile Asn Gln
Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95Ile Ser Arg Leu Glu
Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110Ser Phe Arg
Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125Glu
Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135
140Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser
Val145 150 155 160Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
Arg Asp Val Ser 165 170 175Val Phe Gly Gln Arg Trp Gly Phe Asp Ala
Ala Thr Ile Asn Ser Arg 180 185 190Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp Tyr Ala Val 195 200 205Arg Trp Tyr Asn Thr Gly
Leu Glu Arg Val Trp Gly Pro Asp Ser Arg 210 215 220Asp Trp Val Arg
Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val225 230 235 240Leu
Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250
255Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Met Ala Gln Arg
Ile Glu 275 280 285Gln Asn Ile Arg Gln Pro His Leu Met Asp Ile Leu
Asn Arg Ile Thr 290 295 300Ile Tyr Thr Asp Val His Arg Gly Phe Asn
Tyr Trp Ser Gly His Gln305 310 315 320Ile Thr Ala Ser Pro Val Gly
Phe Ser Gly Pro Glu Phe Ala Phe Pro 325 330 335Leu Phe Gly Asn Ala
Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu 340 345 350Thr Gly Leu
Gly Ile Phe Arg Thr Leu Ser Ser Pro Leu Tyr Arg Arg 355 360 365Ile
Ile Leu Gly Ser Gly Pro Asn Asn Gln Glu Leu Phe Val Leu Asp 370 375
380Gly Thr Glu Phe Ser Phe Ala Ser Leu Thr Thr Asn Leu Pro Ser
Thr385 390 395 400Ile Tyr Arg Gln Arg Gly Thr Val Asp Ser Leu Asp
Val Ile Pro Pro 405 410 415Gln Asp Asn Ser Val Pro Pro Arg Ala Gly
Phe Ser His Arg Leu Ser 420 425 430His Val Thr Met Leu Ser Gln Ala
Ala Gly Ala Val Tyr Thr Leu Arg 435 440 445Ala Pro Thr Phe Ser Trp
Gln His Arg Ser Ala Glu Phe Asn Asn Ile 450 455 460Ile Pro Ser Ser
Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr Asn465 470 475 480Leu
Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly 485 490
495Asp Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val
500 505 510Asn Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile
Arg Tyr 515 520 525Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile
Asp Gly Arg Pro 530 535 540Ile Asn Gln Gly Asn Phe Ser Ala Thr Met
Ser Ser Gly Ser Asn Leu545 550 555 560Gln Ser Gly Ser Phe Arg Thr
Val Gly Phe Thr Thr Pro Phe Asn Phe 565 570 575Ser Asn Gly Ser Ser
Val Phe Thr Leu Ser Ala His Val Phe Asn Ser 580 585 590Gly Asn Glu
Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val 595 600
605Thr8609PRTBacillus thuringiensis 8Met Asp Asn Asn Pro Asn Ile
Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15Ser Asn Pro Glu Val
Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30Tyr Thr Pro Ile
Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45Glu Phe Val
Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60Trp Gly
Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile65 70 75
80Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala
Glu 100 105 110Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala
Leu Arg Glu 115 120 125Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser
Ala Leu Thr Thr Ala 130 135 140Ile Pro Leu Phe Ala Val Gln Asn Tyr
Gln Val Pro Leu Leu Ser Val145 150 155 160Tyr Val Gln Ala Ala Asn
Leu His Leu Ser Val Leu Arg Asp Val Ser 165 170 175Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg 180 185 190Tyr Asn
Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr Ala Val 195 200
205Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu
Thr Val225 230 235 240Leu Asp Ile Val Ala Leu Phe Ser Asn Tyr Asp
Ser Arg Arg Tyr Pro 245 250 255Ile Arg Thr Val Ser Gln Leu Thr Arg
Glu Ile Tyr Thr Asn Pro Val 260 265 270Leu Glu Asn Phe Asp Gly Ser
Phe Arg Gly Met Ala Gln Arg Ile Glu 275 280 285Gln Asn Ile Arg Gln
Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300Ile Tyr Thr
Asp Val His Arg Gly Phe Asn Tyr Trp Ser Gly His Gln305 310 315
320Ile Thr Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Ala Phe Pro
325 330 335Leu Phe Gly Asn Ala Gly Asn Ala Ala Pro Pro Val Leu Val
Ser Leu 340 345 350Thr Gly Leu Gly Ile Phe Arg Thr Leu Ser Ser Pro
Leu Tyr Arg Arg 355 360 365Ile Ile Leu Gly Ser Gly Pro Asn Asn Gln
Glu Leu Phe Val Leu Asp 370 375 380Gly Thr Glu Phe Ser Phe Ala Ser
Leu Thr Thr Asn Leu Pro Ser Thr385 390 395 400Ile Tyr Arg Gln Arg
Gly Thr Val Asp Ser Leu Asp Val Ile Pro Pro 405 410 415Gln Asp Asn
Ser Val Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser 420 425 430His
Val Thr Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr Leu Arg 435 440
445Ala Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu Phe Asn Asn Ile
450 455 460Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser
Thr Asn465 470 475 480Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro
Gly Phe Thr Gly Gly 485 490 495Asp Ile Leu Arg Arg Thr Ser Pro Gly
Gln Ile Ser Thr Leu Arg Val 500 505 510Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr Arg Val Arg Ile Arg Tyr 515 520 525Ala Ser Thr Thr Asn
Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro 530 535 540Ile Asn Gln
Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu545 550 555
560Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe
565 570 575Ser Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe
Asn Ser 580 585 590Gly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val
Pro Ala Glu Val 595 600 605Thr912DNAArtificial
SequenceSynthetically generated oligonucleotide 9taaaccatgg ct
12101990DNABacillus thuringiensis 10gagcgggccc aagcttggat
ccgaattctc attaatcagc agcgagtcca gcttgttggt 60ggaggtgaag agggcgtgag
gctttgtgca cgctccatcg tctcctcttg aatgtggcgt 120cagcaaggat
caactcgatc ttatcgatgt agagttcacc agagctgatg gagccggcgc
180cgaagagagg ttgttcgctg atgccaatga tatctgggtt agcacggaag
gagaaagggt 240tagagaagtc ggtatacctg aaggttctac tagtgaggtt
ctctccgatc tccatagtct 300tttgcaatgg catgttaacg ctgacttggc
caccgactcc tgtggaagcg gcaccggtaa 360gcacaatgac tcttgcgtct
ctagaggaag cgtagcggaa cctaagacgg tacctttgtg 420tgataggaga
gttaatgtta acctggaggg acacaaactc accaatggtg tttctacgga
480ggatatcgcc accggtgaat ccgggccctt taatgacgga ggtccctcca
cccaagcgga 540agcccttgac aagggggatt tgtgtaatga tatcgggatt
gatagtgttt gtgttggtgg 600ctgagtggtg agtccacacg aaggtgggca
aactagtgat attagtgtta tagagagacc 660tggtcaaagt aacgtggcta
agacggtgac tgtatccgac gagtgagttc tcaccgtcaa 720ttggaagttc
gttcaaggag tccactgtgc ctctagacct gtagagcacc tcagcattgt
780ttggctggat gaaacccact ccctggacaa cgttgatccc aagggtaggg
gtgatgttct 840cagatctcct gaagaatgga tttgacaatg tgcggtagaa
gaggttcaac ccgggaaagg 900tacttggagc aatggtacgt cttggctctg
cgttggcagt aattccgtat tgaggggttg 960tgatcacttg ggaactaccg
gtaaagtgag aggtgaccct atggccagcc cagtagtgaa 1020cacctctgat
aaggtctgta tcgatagtca agttattgag gaagtccata aggtgtggag
1080atctgatagc gctgttttca atgttctcga aggagggacc cacacgatag
tcggtaatgt 1140tgatcacagg atcggtgtag acctccctgg tgagttgaga
actagttgga ataggataaa 1200gtctagagtc gtagtttctg aagaaggaaa
tgatgtccaa cacgctaatg gtgagctcac 1260gcctaaactg gttgaagcga
gcccaattcc tcaatccgcc ggtacgtggg agtctgtcaa 1320gcccagtatt
gtaccatcta acagcgtagt cagtgtagat aggaatcaac cttgtgaggt
1380cgttgtaacg ggagttgata gtagcgatat cgaagcccca ggcttgacca
aagacggaca 1440cgtcgcgaag gacagaaagg tgcaagtttg cagcttgaac
atagacggag aggaatggca 1500cttggtagtt ctgcactgag aagagaggaa
tggcggtgac caagatagag ttcatatcgt 1560tgaattgagt acgcatctct
tccttcaatg ctggattagt agggtcagct tcccactctc 1620tgaaggcctc
tgtatagatt ccgtaaaggg
aagagatccc ctcgagacgg ctaattgcct 1680ggttcctagc aaactcttcg
atcttctggt caatgagcaa ctcgatctgt tcaaggaaga 1740gatcccattg
tgatggtccg atggaacccc aaatggcgtc gaacaatcca aggaggatgc
1800caccaatggg agttgcgcta gcgagcaatc tggaaatctc aagagcaatg
ttggtggcca 1860cggtagaatt ggatctttcg atgtccaaga tctcgttctc
tgggttgttg aggcaattgt 1920aagggacgca ctggttctgg ttgttaacga
tagccatggt ttaaagcttg gatcccccgg 1980ggcatgcggg 1990
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