JP2002335967A - New bacterium, gene encoding insecticidal protein, and protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new bacterium having insecticidal activities, a gene encoding an insecticidal protein, and the insecticidal protein. SOLUTION: The new bacterium, Bacillus thuringiensis TK-E6 having the insecticidal activities is provided. Three genes encoding the protein isolated from the bacterium and having the insecticidal activities, and the protein expressed by the genes are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel insecticidal bacterium, a gene encoding an insecticidal protein, and an insecticidal protein.

[0002]

2. Description of the Related Art As the temperature rises due to global warming and the urban greenhouse effect, the area of growth of sanitary pests such as flies and mosquitoes is expanding year by year. As a result, mosquito-borne diseases, such as malaria, which had been endemic to the tropics, are now spreading to temperate zones, and may cause serious damage worldwide in the near future. Therefore, it is urgently necessary to establish an effective method for controlling sanitary pests including dipteran insects. Chemical pesticides are widely used for controlling pests because they are inexpensive and have high immediate effects, but their secondary and tertiary effects on the living environment of other organisms and humans have become a problem. In contrast, bacteria,
For example, pest control using Bacillus thuringiensis (Bt) has high selective activity against target insects, has no harm to humans or livestock, and has no effect on the growth of crops. Active use is receiving worldwide attention. Bacillus thuringiensis (Bt) is a Gram-positive bacterium that widely inhabits soil, and produces granules containing a crystalline insecticidal protein (Cry protein) called a crystal during spore formation. The insecticidal activity of the Cry protein depends on its type, for example, C
ry1 exhibits high insecticidal specificity, such as Lepidoptera (Streptomyces, Kemushi, etc.), and Cry4 exhibits dipterans (Bowgrass, Maggot).

[0003]

[Problems to be solved by the invention] Bacillus thuringiens
The is subspecies israelensis ( Bti ) has the insecticidal activity specific to dipteran insects and is the only fungus that has been put to practical use for controlling mosquitoes. However, it has been known from previous examples that long-term use of one type of Bt promotes emergence of resistant insects, and the emergence of resistant insects against Bti is also considered to be a matter of time. Therefore, it is necessary to find a bacterium having a dipteran insect-specific insecticidal activity in place of Bti. There is also a need for proteins with higher insecticidal activity.

[0004]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have made intensive studies. As a result, a novel bacterium producing an insecticidal protein was found in soil. This bacterium belongs to Bacillus thu ringiensis , Bacillus thu
It was named ringiensis TK-E6. Furthermore, three genes encoding insecticidal proteins were successfully cloned from the bacterial genes.

Accordingly, the present invention provides a novel bacterium, Bacillus.
thuringiensis TK-E6.

[0006] The present invention also relates to SEQ ID NO: 1
It relates to a gene (cryE6L) containing the 973rd nucleotide sequence. The present invention also relates to a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 (CryE6L). The present invention also relates to a protein comprising the amino acid sequence of SEQ ID NO: 2 (CryE6L).

[0007] The present invention also relates to 91-39 of SEQ ID NO: 3.
It relates to a gene (cryE6S) containing the 33rd nucleotide sequence. The present invention also relates to a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 4 (CryE6S). The present invention also relates to a protein comprising the amino acid sequence of SEQ ID NO: 4 (CryE6S).

[0008] The present invention also relates to SEQ ID NO: 5
It relates to a gene (cryE6Q) containing the 332nd nucleotide sequence. The present invention also relates to a gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 6 (CryE6Q). The present invention also relates to a protein comprising the amino acid sequence of SEQ ID NO: 6 (CryE6Q).

The bacterium Bacillus thuringiensis TK-
E6 has the following characteristics. (A) Morphological properties (1) Shape and size of cells: Bacteria (1.0 × 4.0 μm)
m) Chain-like (2) Motility: Yes Flagellated state: Perihair (3) Spores: Yes Spore morphology: semi-rigid, oval spores, no change in bacterial cells by spores

(B) Cultural properties (1) Liquid broth culture: sedimentation (2) Litmus milk: coagulation

(C) Physiological properties (1) Gram stainability: + (2) Reduction of nitrate:-(3) Denitrification reaction: + (4) MR test: + (5) Formation of indole:-(6) ) Production of hydrogen sulfide:-(7) Hydrolysis of starch: + (8) Utilization of citric acid Koser medium:-, Christensen medium: + (9) Utilization of inorganic nitrogen source Nitrate: + w, ammonium salt: + w (10) Urease:-(11) Oxidase:-(12) Catalase: + (13) Growth range pH: pH 5.7 +; pH 6.8 + Temperature: 5 ° C-; 10 ° C +; 30 ° C +;
55 ° C. −; 65 ° C. − (14) Anaerobic culture: + (15) Casein hydrolysis: + (16) Tyrosine agar: − (17) Egg yolk medium: − (18) NaCl: 2 % +; 5% +; 7%
+; 10%-(19) Decomposition of hippurate:-(20) Deamination of phenylalanine:-(21) Requirement of NaCl and KCl:-(22) OF test (Hugh Leifson method):-

(D) Chemical classification properties The sequence of the 5 prime end of the 16S rRNA gene of TK-E6 is shown in SEQ ID NO: 7 (base number: 1548). MicroSeq
The top 10 closely related strains are compared with the database, and their differences are shown below. As a result, TK-E6 was Bacillus thuri
ngiensis .

Table 1 Bacterium name Number of bases Difference (%) Bacillus thuringiensis 1547 0.45% Bacillus cereus 1547 0.74% Bacillus mycoides 1547 0.87% Bacillus flexus 1548 5.07% Bacillus halmapalus 1545 5.15% Bacillus cohnii 1547 5.24% Bacillus simplex 1546 5.47% Bacillus megaterium 1547 5.53% Brevibacterium frigoritolerans 1546 5.69% Bacillus lentus 1547 5.79%

(E) Properties that characterize TK-E6 During the sporulation stage, a crystalline insecticidal protein called crystal is formed in the cells. This bacterium Bacillus thuringiens
is TK-E6 was deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-17981.

Further, the present inventors have succeeded in cloning three genes encoding insecticidal proteins from this fungus. The present invention relates to an isolated gene (cryE6L) containing the 161st A to the 3973th G in the nucleotide sequence of SEQ ID NO: 1. This gene encodes an insecticidal protein. The amino acid sequence corresponding to the start codon ATG at positions 161 to 163 to the stop codon TAG at positions 397 to 973 in the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. The present invention relates to a protein comprising the amino acid sequence of SEQ ID NO: 2 (CryE6L)
Also, an isolated gene encoding Those of skill in the art will appreciate that due to the degeneracy of the genetic code, there may be many genes encoding the amino acid sequence of SEQ ID NO: 2. All such genes are included within the scope of the present invention.

[0015] The present invention relates to an isolated gene (cryE6S) containing the 91st A to the 3933rd A in the nucleotide sequence of SEQ ID NO: 3. This gene encodes an insecticidal protein. From the start codon ATG at positions 91 to 93 in the nucleotide sequence of SEQ ID NO: 3,
The amino acid sequence corresponding to the stop codon TGA at positions 931 to 933 is shown in SEQ ID NO: 4. The present invention also relates to an isolated gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 4 (CryE6S).

The present invention relates to an isolated gene (cryE6Q) containing the 568th A to the 4332th G in the nucleotide sequence of SEQ ID NO: 5. This gene encodes an insecticidal protein. SEQ ID NO: 6 shows the amino acid sequence corresponding to the start codon ATG at positions 568 to 570 to the stop codon TAG at positions 4330 to 4332 in the nucleotide sequence of SEQ ID NO: 5. The present invention relates to a protein comprising the amino acid sequence of SEQ ID NO: 6 (CryE6Q)
Also, an isolated gene encoding

Although the bacterium of the present invention can be used as it is as a microbial pesticide, the gene of the present invention can be used to produce an insecticidal protein encoded by the gene by well-known recombinant DNA technology. The present inventor has proposed recombinant D
The insecticidal proteins (CryE6L, C
ryE6S and CryE6Q) were also successfully expressed. The basic steps for producing the protein of the present invention by the recombinant DNA method are as follows. (1) A DNA that is the gene of the present invention is prepared. (2) The gene is introduced into an expression vector by a method suitable for expressing the protein. (3) Transforming a suitable eukaryotic or prokaryotic host cell with the vector to form a recombinant host cell. (4) culturing the recombinant cells to produce the protein of the present invention. (5) recovering the protein by any suitable method known to those skilled in the art.

The present invention also relates to vectors carrying the nucleic acid molecules according to the invention, in particular plasmids, cosmids, viruses, bacteriophages and other vectors conventionally used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art. For example, Sambrook, Molecular Cloning A LaboratoryMan
ual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology,
Green Publishing Associates and Wiley Interscienc
e, NY (1989), see (1994). Plasmids and vectors preferably used according to the present invention include those well known to those skilled in the art. Alternatively, the genes and vectors of the invention can be reconstituted into liposomes that carry the target cells.

In a preferred embodiment, the nucleic acid molecule present in the vector is linked to a control sequence capable of expressing the gene in prokaryotic or eukaryotic cells. The term "control sequences" refers to regulatory DNA sequences necessary to express the coding sequence to which they bind. The nature of such control sequences will vary with the host organism. In prokaryotes, control sequences generally include a promoter,
Includes ribosome binding site and terminator. In eukaryotes, control sequences generally include promoter, terminator and, in some cases, transactivators or transcription factors. The term "control sequence" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional useful components.

The term "operably linked" refers to positions in a relationship permitting the components to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. It is obvious to those skilled in the art that a double-stranded nucleic acid is preferably used when the control sequence is a promoter.

Therefore, the vector of the present invention is preferably an expression vector. An “expression vector” is a construct that can be used to transform a selected host cell and express a coding sequence in the selected host cell. The expression vector can be, for example, a cloning, binary vector or integrating vector. Expression preferably involves transcription of the nucleic acid molecule into a translatable mRNA. Regulatory elements that ensure expression in prokaryotic and / or eukaryotic cells are well known to those of skill in the art. In the case of eukaryotic cells, they usually include a promoter that ensures initiation of transcription, and optionally a polyA signal that ensures termination of transcription and stabilization of the transcript. Commonly used promoters are the polyubiquitin promoter and, in the case of expression, the actin promoter. Further regulatory elements can include transcription enhancers. Possible regulatory elements enabling expression in prokaryotic host cells are described, for example, in E. coli . E. coli P _L ,
Examples of regulatory elements that are lac, trp or tac promoters and that allow expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast. CMV-, SV40-, RSV- in mammalian and other animal cells
Promoter (Rous sarcoma virus), CMV enhancer, SV40 enhancer or globin intron. Okayama-Berg expression vector pcDV1 (Pharmaci
a), pCDM8, pRc / CMV, pcDNA1, pc
DNA3 (In-vitorogen), pSPORT1 (GIBCO B
Suitable expression vectors such as RL) are known to those skilled in the art.
Another expression system that can be used to express the protein is an insect system. In one such system, Spodoptera fru using Autographa california nuclear polyhedrosis virus (AcNPV) as a vector.
The foreign gene is expressed in giperda cells or Trichoplusia larvae . The coding sequence of the gene of the invention may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. Successful insertion of the coding sequence will render the polyhedrin gene inactive and result in a recombinant virus lacking coat protein. Using the recombinant virus, Spodoptera frugiperda cells or Trichoplusi
a. larva , in which the protein of the invention is expressed (Smith, J. Virol. 46 (1983), 584; Engelha
rd, Proc. Nat.Acad. Sci. USA91 (1994), 3224-322.
7). Advantageously, said vector of the invention comprises a selectable marker.

The invention further relates to a host cell comprising a vector as described above or a gene of the invention, wherein the nucleic acid sequence is foreign to the host cell. By "foreign" the nucleic acid molecule is heterologous with respect to the host cell (meaning it is derived from a cell or organism with a different genetic background) or is homologous with respect to the host cell, but the nucleic acid molecule is Is in a different genetic environment from its naturally occurring counterpart. This means that if the nucleic acid molecule is homologous with respect to the host cell, it is not in its natural location in the genome of the host cell, especially surrounded by different genes. In this case, the nucleic acid molecule may be under the control of its own promoter or under the control of a heterologous promoter. A vector or gene according to the present invention present in a host cell may be integrated into the host cell genome or may be maintained extrachromosomally. In this regard, the genes of the present invention may be used to restore or create mutant genes by homologous recombination (edited by Paszkowski, Homologous Recombi).
nation andGene Sllencing in Plants, Kluwer Academi
c Publishers (1994)).

Accordingly, the present invention relates to host cells containing the vectors or genes of the present invention. Host cells are (arch) bacteria,
It can be any prokaryotic or eukaryotic cell, such as an insect, fungal, plant, or animal cell. Preferred fungal cells include, for example, Sac
cells of the genus charomyces , especially Saccharomyces cerevisiae
Cells.

The term "prokaryotic" is intended to include all bacteria, such as archaebacteria, that can be transformed or transfected with DNA or RNA for expression of the proteins of the present invention. Prokaryotic hosts include, for example, E. coli , St.
yphimurium , Serratia marcescens , and Bacillus su
It can include Gram-positive and Gram-negative bacteria such as btilis . The term "eukaryote" is meant to include yeast, higher plants, insects, and preferably mammalian cells. Depending on the host used in the recombinant production method, the protein encoded by the polynucleotide of the present invention may or may not be glycosylated. The protein of the present invention may or may not have the first methionine amino acid residue. A host can be transformed or transfected with a gene of the present invention using any technique commonly known to those of skill in the art. Furthermore, methods for preparing fused and operably linked genes and expressing them in, for example, mammals and bacteria are well known to those skilled in the art (Sambrook, Molecu
lar Cloning: A Laboratory Manual, Cold Spring Harb
or Laboratory, Cold Spring Harbor, NY, 1989). Hereinafter, the present invention will be described with reference to examples, but it is needless to say that the present invention is not limited to these examples.

[0025]

EXAMPLES Example 1 Design of PCR Primers A novel bacterium of the present invention is a novel bacterium which is prepared by using a degenerated primer designed based on a conserved sequence of a known Cry protein.
R was detected as an amplified DNA fragment was obtained and separated. Several Cry proteins are known, and the N-terminal half has a region containing a relatively conserved amino acid sequence. PCR used to detect bacteria having a gene encoding a Cry protein
Primers were designed as follows. The N-terminal half of the Cry protein has five regions (blocks) containing amino acid sequences relatively conserved in the Cry protein family.
1 to block 5) (see FIG. 1). Cry1Aa1
(Accession No. M11250, Schnepf, HE, Wong, HC,
and Whiteley, HR, "The amino acid sequence of a
crystal protein from Bacillus thuringiensis deduced
from the DNA base sequence ", J. Biol. Chem., 260,
6264-6272 (1985)), Cry4A, Cry4B (Acces
sion No. D00248, D00247 Sen, K., Honda, G., Koyam
a, N., Nishida, M., Neki, A., Sakai, H., Himeno,
M., and Komano, T., "Cloning and nucleotide sequen
ces of the two 130 kDa insecticidal protein genes
of Bacillus thuringiensis var.israelensis ", Agric.
Biol. Chem., 52, 873-878 (1988)), Cry7Aa1.
(Accession No. M64478 Hofte, HR, Annys, K., La
mbert, B., Jansens, S., Soetaert, P., and Peferoen,
M., "Novel Bacillus thuringiensis insecticidal cr
ystal protein with a silent activity against coleo
pteran larvae ", Appl. Environ. Microbiol., 58, 253
6-2542 (1992)) and Cry8Aa1 (Accession No. U
04364 Feitelson, JS, Submitted (15-DEC-1993) to
By comparing the amino acid sequences within the block of the EMBL / GenBank / DDBJ databases. (unpublished)), the primer for the sense strand is a conserved sequence consisting of the eight amino acids in the middle of block 1: Y (V / A) Q (A /
V) Based on the nucleotide sequence corresponding to AN (L / F) (H / N), a 23-mer oligonucleotide having the following nucleotide sequence was synthesized: 5'-TAYGYICARGYIGCIAAYYTHMA-3 '(SEQ ID NO: 8, hereinafter referred to as BtiF4B1). To reduce the degeneracy due to the mixed base, inosine (I) capable of forming a base pair with any of G, A, T and C was inserted at the N position. Similarly, the primer of the antisense strand is a 17-mer oligonucleotide based on the base sequence of the complementary strand of the base sequence corresponding to the sequence consisting of 6 amino acid residues in the latter half of block 3: G (F / H) TGGD : 5'-ATCYCCHCCYGTAWRHC-3 '(SEQ ID NO: 9, hereinafter referred to as BtiR5B3).

Example 2 Primary Screening of Bacteria As a primary screening of bacteria, soil bacteria from which an amplified DNA fragment was obtained by PCR using the primers of Example 1 were isolated. Sterile water was added to the soil sample and stirred to extract cells and spores. This extract was spread on an agar medium for bacterial culture, and cultured at 30 ° C. for 18 hours to form a colony. Each of the resulting colonies was subjected to PCR using the primers of Example 1. The PCR at this stage used a direct PCR method in which cells were directly suspended in a reaction solution without extracting DNA from the cells. The composition of the PCR reaction solution was 10 mM Tris-HCl (pH 8.
3), 50 mM KCl, 2.0 mM MgCl ₂ , 2.5 mM dNTPs, 5.0 μM
Primer BtiF4B1 (sense), 5.0 μM Primer Bt
iR5B3 (antisense), 2.5U Taq polymerase (TaKa
Ra Ex Taq). The PCR program is 1) 94
2 minutes at 94 ° C., 30 seconds at 94 ° C., 3 seconds at 42 ° C.
4) 1 minute at 72 ° C, 5) Step 2 and subsequent steps 29 times, 6) 72
C. for 5 minutes. The PCR product was subjected to 1% agarose gel electrophoresis, and cells from a colony where a fragment of the expected length (about 1 kbp) was obtained were suspended in LB / 15% glycerol and stored at -80 ° C.

[0027] The whole genome containing the plasmid from the soil bacterium obtained in the secondary screening primary screening in Example 3 bacterial extracts and purified, PCR was performed using the same primers as used in the primary screening. The nucleotide sequence of the PCR product was determined, and the amino acid sequence was deduced. Based on the homology between this amino acid sequence and the known Cry protein,
Strains that were predicted to be the gene encoding the protein were selected. A colony of each soil bacterium obtained by the primary screening in Example 1 was inoculated into 5 ml of LB medium, and cultured at 30 ° C. for 18 hours with shaking with monode. The cells were collected by centrifugation, the cells were suspended in a lysozyme buffer, treated with lysozyme at 30 ° C. for 3 hours, and then the plasmid was extracted and purified using an alkaline method. In this method, in addition to the plasmid, a small amount of genomic DN
Although A was contaminated, it was used as it was as a template DNA for PCR. PCR: 10 ng template DNA, primer concentration Bti
Both F4B1 and BtiR5B3 were used at 5 μM, and the enzyme was used with 0.5 units of ExTaq. The reaction was performed at denaturation at 96 ° C for 30 seconds, annealing at 42 ° C for 30 seconds, and extension reaction at 72 ° C for 1 minute.
Repeated 30 times. The annealing temperature was set to the lowest Tm of the mixed primer.

The PCR product was subjected to agarose gel electrophoresis (1% Se
aPlaqueGTG (Takara Shuzo), TAE buffer)
A DNA fragment of about 1 kbp was extracted from the gel. The extracted DNA fragment was used as a TA cloning vector pGEM-T easy (Promega)
It was cloned into the above PCR product insertion site and introduced into E. coli DH5α. A plasmid was prepared from this recombinant, and the inserted DN
Primer M13primerM4 for sequencing the base sequence of fragment A
(Takara Shuzo) and M13primerRV (Takara Shuzo), sequence reaction kit ABI PRISM BigDye Terminator Cycle Sequen
Determined using cing FS Ready Reaction Kit (Applied Biosystems Japan). In this screening method, the target DNA fragment becomes a part of the ORF of the cry gene. Therefore, the obtained nucleotide sequence was replaced with an amino acid sequence, and a homology search was performed for a protein data bank.

Among the isolated strains, a DNA fragment having three different nucleotide sequences was obtained from the strain named TK-E6 by degenerate PCR, and all of them encode an amino acid sequence highly homologous to the known Cry protein. Was. From these results, it was inferred that TK-E6 had three types of genes (cryE6L, cryE6S, cryE6Q) highly homologous to known cry genes.

This bacterium is a spore-forming gram-positive bacterium belonging to Bacillus thuringiensis , and its taxonomic properties are as described above (the bacterium was identified by NCIM Japan, Inc.). T).
This bacterium was named Bacillus thuringiensis TK-E6 and was deposited with the National Institute of Biotechnology and Industrial Technology under the accession number FERM P-17981.

Example 4 Insecticidal Activity of TK-E6 against Bow Fra 1 ml of an overnight culture of TK-E6 was added to a Schaeffer medium (8%
Bouillon, 2 mM MgSO ₄ , 26 mM KCl, 1 mM Ca (NO ₃ ) ₂ , 10
μM MnCl ₂ , 1 μM FeSO ₄ ).
The cells were cultured with shaking for 6 hours. After 96 hours, most of the bacteria formed spores and crystals and autolysed. After confirming complete lysis by microscopic observation, centrifugation (10,000
xg, 4 ° C, 15 minutes) to recover crystals (including spores and cell debris). The pellet was suspended in an equal volume of sterile water and stored at -20 ° C until used for Bow Fra assay. One mosquito larva (3rd instar) and 100 μl of Milli-Q water were placed in each well of a 96-well microtiter plate. 100 μl of the sample solution appropriately diluted was added, and 25
It left at ° C. After 18 hours, the number of dead insects was counted. Table 2 shows the results.

[Table 2] Sample Concentration Number of Insects Insect rate (x10 ⁴ spores / ml) (out of 48) (%) 100 48 100 50 38 79.2 25 19 39.6 10 6 12.5 5.0 2 4.2 500.00.00.00.0

Example 5 Cloning of cryE6L The entire nucleotide sequence of cryE6L of TK-E6 was determined by the procedure of genome walking. Based on the base sequence of the about 1 kb DNA fragment of cryE6L obtained in the secondary screening of Example 3, a sequence primer was synthesized,
The sequence was performed using 1 μg of DNA prepared as in the case of CR as a template. The sequence was repeated by synthesizing the next sequence primer from the newly determined nucleotide sequence, and the nucleotide sequence of a DNA region of about 5 kbp including the ORF of the CryE6L protein was determined. next,
PCR was performed using two primers positioned so as to sandwich the ORF: 5'-CACTGCAAGATCATGGCTGTCAC-3 '(E6LcryS1, SEQ ID NO: 10) 5'-AATTAGCAGGCTTAGCACAGACTTA-3' (E6LcryA2, SEQ ID NO: 11). PGEM-T easy
The primer used for genome walking and the universal primer M13prime
Determine the nucleotide sequence using rM4, M13primerRV, 4226 b
It was confirmed that the nucleotide sequence of this DNA fragment consisting of p (SEQ ID NO: 1) matched the result of genome walking.
In the determined 4226 bp DNA fragment (SEQ ID NO: 1), starting from the start codon at positions 161 to 163 and the stop codon at positions 3971 to 3973, assuming that the base at the 5 'end of this base sequence is No. 1, The last 3813 base coding region can be taken. This ORF has a molecular weight of 14
It was presumed to encode a 3 kDa protein (SEQ ID NO: 2). In the N-terminal half of this protein, a region conserved by Cry protein (block 1 to block 5)
And the amino acid sequence at the C-terminal side had about 40% homology with other known Cry proteins.

Example 6 Cloning of cryE6S Of the three types of cry genes possessed by TK-E6, the entire nucleotide sequence of a gene named cryE6S was subjected to genome walking and reverse
It was determined by the PCR method (Inverse PCR). Based on the nucleotide sequence of the cryE6S about 1 kb DNA fragment obtained in the secondary screening, a sequence primer was synthesized, and a nucleotide sequence of about 800 bp upstream from the primer BtiF4B1 and about 500 bp downstream from the primer BtiR5B3 were synthesized in the same way as in genome walking. Were determined. Antisense primer E6SinvA1 in the upstream part of the obtained base sequence, sense primer E6Sinv in the downstream part
Synthesize S1 and reverse PCR method (Naoki Ogasawara, Yasuhiro Kasahara: Cell Engineering Separate Volume Tips Series Revised PCR Tips (Supervised by Toshiharu Maki)
p.61-64, 1999, Shujunsha), the undetermined portion was determined in the N-terminal and C-terminal nucleotide sequences of cryE6S.

Preparation of template DNA: In the same manner as in the secondary screening, 10 μg of the plasmid and genomic DNA prepared from TK-E6 were completely digested with the restriction enzyme EcoRI. After purifying the DNA fragment by phenol treatment and ethanol precipitation, the DNA fragment was dissolved in 500 μl of sterilized water to a concentration of 20 μg / ml. To this solution, add 2 units of T4 DNA ligase (TaKaRa), 500 μl of 10 × ligation buffer, and sterile water to make a total volume of 5 ml (D
The final concentration of NA was 2 μg / ml), and the reaction was performed at 16 ° C for 18 hours.
Self-cyclization of the I fragment was performed. After the ligation reaction,
The DNA solution was concentrated with 2-butanol, and the DNA was purified by 2-propanol precipitation. Then, add sterile water and add 100 μg / m
This solution was used as a template DNA in the following inverse PCR reaction.

Reverse PCR: Reverse PCR used TaKaRa LA Taq to amplify 5-10 kbp. The primer was designed to have a Tm value of 65 ° C. or higher so that the PCR program could be performed in two steps. The following inverse primers were used. E6SinvA1 (38-66) 29mer: 5'-TCTTGACTGATCGCCCCTGCCACCATTAT-3 '(SEQ ID NO: 12) E6SinvS1 (1751-1780) 30mer: 5'-TCGGTGGAGGCGTAGAGGAATCGGAGAGCA-3' (SEQ ID NO: 13) The reaction solution composition (50 μl) is as follows. Appears; 200 ng template DN
A, 0.2 μM E6SinvA1, 0.2 μM E6SinvS1, LA Taq 2.5
U, 10 mM dNTPs (2.5 mM each), 2.5 mM MgCl ₂ . As the buffer, the one attached to the enzyme kit (without containing Mg ²⁺ ) was used.
The PCR program (30 cycles) is as follows.
Denaturation 94 ° C 30 seconds, annealing and extension reaction 65 ° C
10 minutes.

The PCR product was subjected to agarose electrophoresis (1% SeaPl
aque GTG, TAE buffer), about 7.5 kbp
Since a single amplification product was detected in the above, it was extracted and purified, and cloned into a TA cloning vector pGEM-T easy. By sequencing this plasmid with the sequencing primers M13 Primer M4 and M13 Primer RV, it was confirmed that the inserted 7.5 kbp DNA fragment was amplified by the inverse primers E6SinvA1 and E6SinvS1. In addition, this plasmid is
When digested with I, the inserted DNA fragment is about 3 kbp and about 4.5 kbp.
bp. From the above, it was determined that the cloned DNA fragment was an inverse PCR product using a self-closing EcoRI fragment containing cryE6S as a template. Therefore, about 100 bp upstream of E6SinvA1 and E6S
The nucleotide sequence of about 2.5 kbp downstream of invS1 was determined. By combining this result with the results of degenerate PCR and genome walking, a 3933 bp fragment containing the ORF of the CryE6S protein was obtained.
The nucleotide sequence of the DNA region was determined.

The determined 3933 bp DNA fragment (SEQ ID NO: 3)
If the base at the 5 'end of this nucleotide sequence is No. 1,
A 3843 base coding region can be taken, beginning at the start codon at positions 91-93 and ending at the stop codon at positions 3931-3933. This ORF was presumed to encode a protein (CryE6S) consisting of 1280 amino acids and having a molecular weight of 144 kDa (SEQ ID NO: 4). Also, 7 from the start codon
The consensus sequence (GGAGG) of the ribosome binding site can be taken upstream (79th to 83rd) of the base.
The third stop codon (TAG) can be taken in the same frame (reading frame) as the CryE6S ORF.

The nucleotide sequence of ORF was confirmed by determining the nucleotide sequence of a PCR product using two primers designed to sandwich the ORF. The two primers are based on the base sequence (underlined) of the complementary strand of 25 bases upstream including the codon (GTA) of the amino acid Val at the C-terminal, and the antisense strand includes the base 40 nucleotides downstream from the initiation codon for the sense strand. The primers were designed and a recognition sequence (CCATGG) for the restriction enzyme NcoI was ligated to the 5 'side of each primer. Sense strand primer: NcoE6SNM (48 mer) 5'-CCATGGGT ATGAAACAAAATTACAATGATGAATATGACATTATAGAT
A -3 '(SEQ ID NO: 14) Antisense strand primer: NcoE6SCt (32 mer) 5'-CCATGGT TACATTGGTAGGTGGCATGATCGGT -3' (SEQ ID NO: 1)
5) After PCR, a product of about 3.8 kbp was cloned into pGEM-T easy, and it was confirmed that the nucleotide sequence was consistent with the results obtained by genome walking and reverse PCR. In the N-terminal half of CryE6S, a region conserved by Cry protein (block 1)
~ Block 5), and the amino acid sequence of the C-terminal half had about 40% homology with other known Cry proteins.

Example 7 Cloning of cryE6Q Of the three types of cry genes of TK-E6, the entire nucleotide sequence of a gene named cryE6Q was subjected to genome walking and reverse
It was determined by the PCR method (Inverse PCR). A sequence primer was synthesized based on the base sequence of the about 1 kb DNA fragment of cryE6Q obtained in the secondary screening, and a base sequence of about 1.2 kbp upstream from the primer BtiF4B1 was determined by the procedure of genome walking, and the 5 ′ region of cryE6Q was determined. Detected. Of the newly determined base sequence, the antisense primer E6Qi based on the base sequence of the untranslated region corresponding to the upstream portion of cryE6Q
Design nvA1 and sense primer E6QinvS1, reverse PCR
The undetermined part was determined from the nucleotide sequence of the 3 'region of cryE6Q by the method.

Preparation of Template DNA As in the case of cryE6S, 10 μg of the plasmid and genomic DNA prepared from TK-E6 were completely digested with the restriction enzyme XbaI, and self-cyclized by the ligation reaction was used as the template DN.
Used as A.

The following inverse PCR inverse primers were used. E6QinvA1 (32 mer) 5'-GGATGGTTTTTCTGCGCTTTTCTTCTATGACA -3 (SEQ ID NO: 1
6) E6QinvS1 (36mer) 5'-AAGTATTTGCCTAGATAATGTGCTGAAATGTGGTGT-3 '(SEQ ID NO: 17) The composition of the reaction solution (50μ) is as follows: 200 ng template DN
A, 0.2 μM E6QinvA1, 0.2 μM E6QinvS1, LA Taq 2.5
U, 10 mM dNTPs (2.5 mM each), 2.5 mM MgCl ₂ . As the buffer, the one attached to the enzyme kit (without containing Mg ²⁺ ) was used.
The PCR program (30 cycles) is as follows: denaturation 94 ° C for 30 seconds, annealing and extension reaction 65 ° C
10 minutes.

The PCR product was subjected to agarose electrophoresis (1% SeaPl
aque GTG, TAE buffer), about 7.3 kbp
Since a single amplification product was detected in the above, it was extracted and purified, and cloned into a TA cloning vector pGEM-T easy. By sequencing this plasmid with the sequencing primers M13 Primer M4 and M13 Primer RV, it was confirmed that the inserted 7.3 kbp DNA fragment was amplified by the inverse primers E6QinvA1 and E6QinvS1. In addition, this plasmid was replaced with the restriction enzyme XbaI.
When digested with, the inserted DNA fragment is about 1.1 kbp and about 6.2 kbp.
It was divided into two kbp. Based on the above, it was determined that the cloned DNA fragment was an inverse PCR product using a self-closing XbaI fragment containing cryE6Q as a template. Therefore,
Approximately 150 bp upstream of E6QinvA1 and E
The nucleotide sequence of about 4 kbp downstream of 6QinvS1 was determined. By combining this result with the results of degenerate PCR and genome walking, a 4332 bp fragment containing the ORF of the CryE6Q protein was obtained.
The nucleotide sequence of the DNA region was determined.

The determined 4332 bp DNA fragment (SEQ ID NO: 5)
If the base at the 5 'end of this nucleotide sequence is No. 1,
A coding region of 3765 bases can be taken, beginning at the start codon at positions 568-570 and ending at the stop codon at positions 4330-4332. This ORF is a protein (CryE6Q) consisting of 1254 amino acids and having a molecular weight of 143 kDa (SEQ ID NO: 6)
Was supposed to code. In addition, a consensus sequence (GGAGG) of a ribosome binding site can be obtained 7 bases upstream from the start codon (positions 556 to 560). The 520th to 522nd stop codon (TGA) is the ORF of CryE6Q
In the same frame (reading frame) as.

The nucleotide sequence of the ORF was confirmed by determining the nucleotide sequence of a PCR product performed using two primers designed to sandwich the ORF. The sense strand primer was designed based on the base sequence (underlined) of the 38 bases downstream including the initiation codon and the antisense strand primer including the C-terminal amino acid Thr codon (ACA) and the 28 bases upstream of the complementary strand. A recognition sequence (GCATGC) for the restriction enzyme SphI was ligated to the 5 'side of each primer. Sense strand primer: SphE6QNM (46 mer) 5'-GCATGCAG ATGAATTCAAATTATAACAATGAACATGAGATCTTAGA-
3 '(SEQ ID NO: 18) Antisense strand primer: SphE6QCt (35 mer) 5'-GCATGCT TGTTAGATCCTGTTCTAAGTTTCCCGCC -3' (SEQ ID NO: 19) After PCR, a product of about 3.8 kbp was cloned into pGEM-T easy, and the nucleotide sequence was changed. The results were confirmed to be consistent with those obtained by genome walking and reverse PCR. The N-terminal half of CryE6Q contains regions conserved by the Cry protein (block 1 to block 5), and the amino acid sequence of the C-terminal half has about 40% homology with other known Cry proteins. I was

Example 8 Expression of cryE6L in E. coli A His-tag fusion protein in which six histidine residues were added to the C-terminal of CryE6L was prepared using an E. coli expression system, and confirmed by Western blotting. The used expression vector pQE-70 (QIAGEN) has a recognition sequence (GCATGC) for the restriction enzyme SphI as a foreign gene insertion site, and uses ATG in the sequence as a start codon. Upstream of the insertion site is a T5 promoter, two lac operators, and a ribosome binding site, which can induce the expression of a foreign gene by IPTG. In addition, a 6 × His tag is arranged downstream, and the reading frame is aligned with the 3′-end of the inserted gene, thereby adding a histidine residue to the C-terminus of the His-ta tag.
The product can be obtained as a g-fusion protein.

Preparation of cryE6L Amino Acid Coding Region From the start codon of cryE6L to the base immediately before the stop codon was amplified by PCR. The sense strand primer SphE6LNM has a sequence of 36 bases (underlined) downstream from the start codon and its 5 ′
On the-side, two bases (A
G) and a recognition sequence for restriction enzyme SphI (GCATGC). The antisense strand primer SphE6LCt contains the complementary strand sequence (underlined) of 32 bases upstream from the codon (TTA) of the C-terminal amino acid of the CryE6L protein, and the 6xHis tag and reading frame of the expression vector on the 5'-side. One base (T) for matching and a recognition sequence of restriction enzyme SphI (GCATGC) were ligated. Sense strand primer: SphE6LNM (44mer) 5'-GCATGCAG ATGAACCAAAATGACAACAAGAATGAATATGAAATA- 3 '(SEQ ID NO: 20) Antisense strand primer: SphE6LCt (39mer) 5'-GCATGCT TAAGTCGTCTTCCATCTGTTCCATACAAATGA- 3' (SEQ ID NO: 21) ) Is 5 ng template DNA (same as for secondary screening), 0.2 μM SphE6LNM, 0.2 μM SphE6L
Ct, LA Taq 2.5 U, 10 mM dNTPs (2.5 mM each), 2.5 mM
MgCl ₂ . As the buffer, the one provided with the enzyme kit (without Mg ²⁺ ) was used. The PCR program was 30 cycles of denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, and extension reaction at 72 ° C for 5 minutes. After PCR, a product of about 3.8 kbp was cloned into pGEM-T easy, the nucleotide sequence was determined using the sequencing primers used in Example 5, and the inserted fragments were SphE6LNM and SphE6LCt.
It was confirmed that it was the amino acid coding region of cryE6L amplified in step 1. Next, 5 μg of this plasmid was added to the restriction enzyme SphI.
(50 U, 37 ° C, 2 hours) and agarose electrophoresis (1% SeaPlaqueGTG, TAE buffer) to ca.
The yE6L fragment was separated and purified.

Cloning of cryE6L into Expression Vector An approximately 3.8 kbp cryE6L fragment digested with SphI was digested with the expression vector pQ.
It was cloned into the SphI site of E-70, the orientation of the insert was the same as the transcription direction of the T5 promoter of the vector, and the reading frame of cryE6L was the initiation codon of the vector and 6 × H
The agreement with the reading frame of the is tag was confirmed by nucleotide sequencing with universal primers pQEprimerPR and pQEprimerRV. This recombinant plasmid was named pQE70-cryE6L, and Escherichia coli M15 was used to induce the expression of CryE6L protein.
[pREP4] (QIAGEN).

Induction of Expression of CryE6L-His Tag Fusion Protein Escherichia coli M15 [pREP4] having pQE70-cryE6L was added to TB medium (500 μg).
/ ml carbenicillin, 25 μg / ml kanamycin, and 1% glucose) were inoculated into 50 ml, and cultured with shaking at 37 ° C. overnight. After centrifugation, the cells were suspended in 50 ml of a fresh TB medium (containing 1 mM IPTG), and cultured with shaking at 37 ° C. overnight to induce the expression of the CryE6L-His tag fusion protein.

Purification and CryE6L-His tag fusion protein
After induction with fine check IPTG, harvested medium was centrifuged, the cells
The suspension was suspended in 10 ml of Lysis Buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole). This solution was sonicated to give a cell lysate, and centrifuged (15000 rpm, 2 minutes) to precipitate cell debris. Ni-NT for 4 ml of collected supernatant
A Agarose (QIAGEN) was added at a rate of 1 ml, and the mixture was gently stirred at 4 ° C. for 1 hour to allow the His-tagged protein to adsorb to the resin. Then, the resin was packed in a polypropylene column. The resin is Wash Buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 20 m
M imidazole) 2 times with 4 ml, Elution Buffe
The His-tag fusion protein was eluted by passing 0.5 ml of r (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole) four times to obtain a purified His-tag fusion protein. When the purified His-tag fusion protein (1 μg) was electrophoresed on SDS-PAGE (8% polyacrylamide gel) and subjected to CBB staining, a band was detected at about 160 kDa. This band was subjected to Western blotting (primary antibody: Tetra / His ^™ Antibody, secondary antibody: Peroxidase-Conjugated Rabbit Anti-Mouse Immu
noglobulins (QIAGEN)).
It was confirmed that the protein was a 6L-His tag fusion protein (FIG. 2).

Example 9 Expression of cryE6S in E. coli pGEM-T easy (Example 6) obtained by cloning a PCR product using a sense strand primer: NcoE6SNM (SEQ ID NO: 14) and an antisense strand primer: NcoE6SCt (SEQ ID NO: 15) ) Was digested with the restriction enzyme NcoI, and the obtained approximately 3.8 kbp fragment was cloned into the NcoI site of the expression vector pQE-60 (QIAGEN) and named pQE60-cryE6S. pQE-60 has an NcoI recognition sequence (CCATGG) as a foreign gene insertion site, and ATG in the sequence
Is used as the start codon. Others are the same as pQE-70. Confirmation of insert orientation and reading frame, induction of expression of CryE6S-His tag fusion protein, purification and confirmation is cryE6L
Performed in the same manner as

Example 10 Expression of cryE6Q in Escherichia coli pGEM-T easy (Example 7) obtained by cloning PCR products using a sense strand primer: SphE6QNM (SEQ ID NO: 18) and an antisense strand primer: SphE6QCt (SEQ ID NO: 19) ) Was digested with the restriction enzyme NcoI, and the obtained approximately 3.8 kbp fragment was cloned into the SphI site of the expression vector pQE-70 (QIAGEN) and named pQE70-cryE6Q. Confirmation of orientation and reading frame of insert, induction of expression of CryE6Q-His tag fusion protein,
Purification and confirmation were performed as for cryE6L.

[0052]

[Sequence Listing] Sequence Listing <110> Kinki University <120> New Bacteria and Gene Encoding Insecticidal Protein <130> 172148 <160> 19 <210> 1 <211> 4226 <212> DNA <213> Bacillus thuringiensis <400> 1 cactgcaaga tcatggctgt cacatgcttc aatttacggg aacgattgaa tgtaaacaca 60 ttccacatac ataaaacacc agacagctat ctgtattcgt agcatataca agcgaccaca 120 actgttacga cacataagaa aaatacgagg aggtttatac atgaaccaaa atgacaacaa 180 gaatgaatat gaaatattgg atagtggaaa tttgtcttat caaccaaggt atccattagc 240 gaatgctcca ggttctaaac tgcaaaatat gggttataaa gaatggaggg atatgtgtcc 300 agatgcaaaa agacagcttc tcaaacagat aggaattagc ccgtttgagc ctgatgaatg 360 gaccaaggta ggacagtctt taacgatatc ggcggggcta gttgctgcaa tagcaggtgt 420 ggcttctgtt gcctttccac cattagcaat cgtcgcaggc gcattcgcaa ttatttctat 480 gttcttcgat gtgttatggc cggaatctga aaataatact aattcgcaag tcgtatgggc 540 ggatttcgcc gctgcagcag aagaaatgat ggatgaaaaa attgcagatg agataaaagc 600 ggaagctgtt cttcaattaa gaattgtaca gtcaagacta agagattatc aacaggcagc 660 ctgtaatttc caaagcgatc ctaataa cga aagttataag gcgttattaa gagatgcatt 720 tgatgatgca gatgatgcac ttaaagaagt catgatattg tttagtagag aaggttacga 780 acaactactg ttattcgatt acgtgcaggc tgccaacctt catctacttt tattaaagga 840 tgttgtacga tttggagttg ggtggggatt tccaccgggc agggtggagc aatattattc 900 gaacccgaca aacctaggta acccaggcat ggtacaatta ttagcaaaat acacaaatta 960 tagtacgagc ctatgttgga aagggataga ggaaagaaag tggatggtcg aaagcgaata 1020 tcgcagcaac aatgaggagt actatgcgta tcgtagcaat atgacgatga tggtgttaga 1080 tatggttgca ctttggccaa catatgatcc agtcaagtat ccatatgcta cagctgtaga 1140 actcacgcga gaaatatatt ctcttatagc aggtgggttc cgtgactata aaggttatat 1200 gcctattcag tacacatggg agaagcctgg tagtttagtt acaaatttag agcgattcac 1260 tatttatacg tggaaaaact tcgagtattt tccattcgct ggggtagaaa caacctatca 1320 gaccattggg actgggtcta gtagtacgaa acaggcggga gtgattccaa gccctgaaga 1380 ggggactgcg tggacaactc ctgggataaa tgtagagtat catttggtgg agagtttcat 1440 atacggtaca ctaacccaac tgctgttcta tgatgtatac aacacacctg cattcaaagc 1500 cggatcggac gcagtagaag ttcccggatc catcgc tgga acaccgtgta aaaatatacc 1560 tctagacgcg aacgatgtta atgtttgtgt accaactcta tgggaggagt ctcctgctaa 1620 tcctgaaggg gtttgttatc cctacaatcg ggagatgcag agtaatctat tatcggaagt 1680 tatcccagag gatcctaagc tactcacact ccctcacgta cctccattag gatatataga 1740 tgcgttcgct ttcgcttggc gatctacaac gtgcgatact agatacaatc taataccagc 1800 aaacaagatt ggtcaaatcc ccgcagttaa gggcaattgg ttaggacaga gtggttctag 1860 cgtggtaagg acttctggta atacaggggg ggatgtagta agactctatg agttcggaaa 1920 tcttggaatg actgtgagat tctcagaaaa tcgctcatat atcatacgat tacgttatgc 1980 aactgttgca gatgatctca atataattgt tagggtacaa agaagagggg agttagagta 2040 tgaaagtgaa ttaccgctca atcaaacatc aaataattca acaactcagt ggaagtttga 2100 agattatgga tatcaagagg tgggtggttt ctatccacaa gttggggaag agtacgagct 2160 ttggttttct cccgtgggta ctgaactcag tagccatatg gacatcgaca aaatcgaatt 2220 cattccaatg gaaacatctt tggaggaata tctagcaaat caagatatag aaaaggcaag 2280 aaaggccgtg aatgccttgt ttacaggtga tgtgaaaaat gccctgaaat tgaacgtgac 2340 ggattacgca atagatcaag ctgccaattt ggtagaatgt g tatcggagg aattctatgc 2400 tcaagaaaaa atgattctac tggatcaagt gaaagtggcg aaacgactga gtcaagcacg 2460 gaatctattg aactacggag attttgaatc accagagtgg tcaagagaga atggatggaa 2520 aacaagtcgc catgtctctg taagagcgga taacccagtc tttaaaggac gatatctcca 2580 catgccagga gtgacaagtc cgtcattttc taacaatacc tatcctacgt atgtctatca 2640 aaaggtcgat gaatcgaaat taaaatccta tacacggtac ctggtacgcg ggtttgttgg 2700 aaatagtaaa gatctggaac tactggtgga gagatacgga aaagatgtcc atgtggaaat 2760 ggatgtacca catgacattc ggtattcttt acagacgaat gaatgtggtg gcttcgatcg 2820 ctgccgacct gtatcctatc tagctcgctc ctctcatgca tgtacatgta aggataccgc 2880 ttccatgcat acagattgtc agtgtcaaaa caaagcgaat cgtactgtaa cgaacatgta 2940 tacaaacgta tcgccaggta gtgcgatgta taccgatggg tttcatgccc aaaaatcctg 3000 tggatgcaag aacaacgaca tgtatcagaa tggaacacat ccgcataaat cttgtggatg 3060 caaagaccca catgtcttct cgtaccatat tgatacagga tgtgtagatc cagaagaaaa 3120 tcttggtttg tggttcgcct tgaaaattgc gggtgaaaat ggtgtcgcga acatcgacaa 3180 cctggaaatc atcgaggcac aaccactcac aggggaagca ttagcac gtg tgaaaaaacg 3240 cgaacagaga tggaaacaag aaagggataa aaagcggtta gaaacagaga aagctgtaca 3300 aacagcgcaa ggtgcgattc agaatctatt tacaaatacg cagcaaaacc ttctgaaatt 3360 tgaaacgctg ttccctcaaa ttgtcaatgc ggagatgctg gtacaacaga ttccctatgt 3420 gtatcatcca ttcttaagcg gggcacttcc agctgtagca ggtatgaatt ttaaaatcgt 3480 ccaacaacta tcagcaacga ttggaaatgc ccgttcgtta tataatcaac gaaatctcgt 3540 acaaaatggt acatttagtt caggtacagg aagctggcat gtgtcagaag gcgtagaggt 3600 gcagccactt caaaatacat ctgtgcttgt cctatcagaa tggagtcatg aagcgtccca 3660 gcaggtaagc gttgatccag atcgcggata tgtgttacgt gtaactgcgc gaaaagaagg 3720 cgtagggaaa ggcactgtaa cactaagtga ttgtgcagac tatacagaaa cactgacctt 3780 tacatcttgt gattttaaca cgtctggttc ccaaacgatg acaagtggta cattatctgg 3840 atttgtgacg aagacgttag agattttccc agatactgat cgtattcgaa ttgatattgg 3900 ggaaaccgaa ggaacgttta agattgaaag tgtggaactc atttgtatgg aacagatgga 3960 agacgactta taggggtagt gagacggttc gtgctctagt ccgtcagata atttaaataa 4020 catgaaaaaa cctactattt caaagtaggt tcaatttatc taacattttt g ttcgagatg 4080 ggtttgtata cagtaatgta gcatgtatat tagaatgacc cgctatatgt gccacttcat 4140 gtacatcaaa accattttcc agtgcgtgtg aacaaaaata atgtcttaga tcatgtggtt 4200 ttaagtctgt gctaagcctg ctaatt 4226

<210> 2 <211> 1270 <212> PRT <213> Bacillus thuringiensis <400> 2 Met Asn Gln Asn Asp Asn Lys Asn Glu Tyr Glu Ile Leu Asp Ser Gly 1 5 10 15 Asn Leu Ser Tyr Gln Pro Arg Tyr Pro Leu Ala Asn Ala Pro Gly Ser 20 25 30 Lys Leu Gln Asn Met Gly Tyr Lys Glu Trp Arg Asp Met Cys Pro Asp 35 40 45 Ala Lys Arg Gln Leu Leu Lys Gln Ile Gly Ile Ser Pro Phe Glu Pro 50 55 60 Asp Glu Trp Thr Lys Val Gly Gln Ser Leu Thr Ile Ser Ala Gly Leu 65 70 75 80 Val Ala Ala Ile Ala Gly Val Ala Ser Val Ala Phe Pro Pro Leu Ala 85 90 95 Ile Val Ala Gly Ala Phe Ala Ile Ile Ser Met Phe Phe Asp Val Leu 100 105 110 Trp Pro Glu Ser Glu Asn Asn Thr Asn Ser Gln Val Val Trp Ala Asp 115 120 125 Phe Ala Ala Ala Ala Glu Glu Met Met Met Asp Glu Lys Ile Ala Asp Glu 130 135 140 Ile Lys Ala Glu Ala Val Leu Gln Leu Arg Ile Val Gln Ser Arg Leu 145 150 155 160 Arg Asp Tyr Gln Gln Ala Ala Cys Asn Phe Gln Ser Asp Pro Asn Asn 165 170 175 Glu Ser Tyr Lys Ala Leu Leu Arg Asp Ala Phe Asp Asp Ala Asp Asp 180 185 190 Ala Leu Lys G lu Val Met Ile Leu Phe Ser Arg Glu Gly Tyr Glu Gln 195 200 205 Leu Leu Leu Phe Asp Tyr Val Gln Ala Ala Asn Leu His Leu Leu Leu 210 215 220 Leu Lys Asp Val Val Arg Phe Gly Val Gly Trp Gly Phe Pro Pro Gly 225 230 235 240 Arg Val Glu Gln Tyr Tyr Ser Asn Pro Thr Asn Leu Gly Asn Pro Gly 245 250 255 Met Val Gln Leu Leu Ala Lys Tyr Thr Asn Tyr Ser Thr Ser Leu Cys 260 265 270 Trp Lys Gly Ile Glu Glu Arg Lys Trp Met Val Glu Ser Glu Tyr Arg 275 280 285 Ser Asn Asn Glu Glu Tyr Tyr Ala Tyr Arg Ser Asn Met Thr Met Met 290 295 300 Val Leu Asp Met Val Ala Leu Trp Pro Thr Tyr Asp Pro Val Lys Tyr 305 310 315 320 Pro Tyr Ala Thr Ala Val Glu Leu Thr Arg Glu Ile Tyr Ser Leu Ile 325 330 335 Ala Gly Gly Phe Arg Asp Tyr Lys Gly Tyr Met Pro Ile Gln Tyr Thr 340 345 350 Trp Glu Lys Pro Gly Ser Leu Val Thr Asn Leu Glu Arg Phe Thr Ile 355 360 365 Tyr Thr Trp Lys Asn Phe Glu Tyr Phe Pro Phe Ala Gly Val Glu Thr 370 375 380 Thr Tyr Gln Thr Ile Gly Thr Gly Ser Ser Ser Thr Thrs Lys Gln Ala Gly 385 390 395 400 Val Ile ProSer Pro Glu Glu Gly Thr Ala Trp Thr Thr Pro Gly Ile 405 410 415 Asn Val Glu Tyr His Leu Val Glu Ser Phe Ile Tyr Gly Thr Leu Thr 420 425 430 Gln Leu Leu Phe Tyr Asp Val Tyr Asn Thr Pro Ala Phe Lys Ala Gly 435 440 445 Ser Asp Ala Val Glu Val Pro Gly Ser Ile Ala Gly Thr Pro Cys Lys 450 455 460 Asn Ile Pro Leu Asp Ala Asn Asp Val Asn Val Cys Val Pro Thr Leu 465 470 475 475 480 Trp Glu Glu Ser Pro Ala Asn Pro Glu Gly Val Cys Tyr Pro Tyr Asn 485 490 495 Arg Glu Met Gln Ser Asn Leu Leu Ser Glu Val Ile Pro Glu Asp Pro 500 505 510 Lys Leu Leu Thr Leu Pro His Val Pro Pro Leu Gly Tyr Ile Asp Ala 515 520 525 Phe Ala Phe Ala Trp Arg Ser Thr Thr Cys Asp Thr Arg Tyr Asn Leu 530 535 540 Ile Pro Ala Asn Lys Ile Gly Gln Ile Pro Ala Val Lys Gly Asn Trp 545 550 555 560 Leu Gly Gln Ser Gly Ser Ser Val Val Arg Thr Ser Gly Asn Thr Gly 565 570 575 Gly Asp Val Val Arg Leu Tyr Glu Phe Gly Asn Leu Gly Met Thr Val 580 585 590 Arg Phe Ser Glu Asn Arg Ser Tyr Ile Ile Arg Leu Arg Tyr Ala Thr 595 600 605 Val Ala Asp Asp Leu Asn Ile Ile Val Arg Val Gln Arg Arg Gly Glu 610 615 620 620 Leu Glu Tyr Glu Ser Glu Leu Pro Leu Asn Gln Thr Ser Asn Asn Ser 625 630 635 640 Thr Thr Gln Trp Lys Phe Glu Asp Tyr Gly Tyr Gln Glu Val Gly Gly 645 650 655 Phe Tyr Pro Gln Val Gly Glu Glu Tyr Glu Leu Trp Phe Ser Pro Val 660 665 670 Gly Thr Glu Leu Ser Ser His Met Asp Ile Asp Lys Ile Glu Phe Ile 675 680 685 Pro Met Glu Thr Ser Leu Glu Glu Tyr Leu Ala Asn Gln Asp Ile Glu 690 695 700 Lys Ala Arg Lys Ala Val Asn Ala Leu Phe Thr Gly Asp Val Lys Asn 705 710 710 720 Ala Leu Lys Leu Asn Val Thr Asp Tyr Ala Ile Asp Gln Ala Ala Asn 725 730 735 Leu Val Glu Cys Val Ser Glu Glu Phe Tyr Ala Gln Glu Lys Met Ile 740 745 750 Leu Leu Asp Gln Val Lys Val Ala Lys Arg Leu Ser Gln Ala Arg Asn 755 760 765 765 Leu Leu Asn Tyr Gly Asp Phe Glu Ser Pro Glu Trp Ser Arg Glu Asn 770 775 780 Gly Trp Lys Thr Ser Arg His Val Ser Val Arg Ala Asp Asn Pro Val 785 790 795 800 Phe Lys Gly Arg Tyr Leu His Met Pro Gly Val Thr Ser Pro Ser Phe 805 810 815 Ser Asn Asn Th r Tyr Pro Thr Tyr Val Tyr Gln Lys Val Asp Glu Ser 820 825 830 Lys Leu Lys Ser Tyr Thr Arg Tyr Leu Val Arg Gly Phe Val Gly Asn 835 840 845 Ser Lys Asp Leu Glu Leu Leu Val Glu Arg Tyr Gly Lys Asp Val His 850 855 860 Val Glu Met Asp Val Pro His Asp Ile Arg Tyr Ser Leu Gln Thr Asn 865 870 875 880 Glu Cys Gly Gly Phe Asp Arg Cys Arg Pro Val Ser Tyr Leu Ala Arg 885 890 895 895 Ser Ser His Ala Cys Thr Cys Lys Asp Thr Ala Ser Met His Thr Asp 900 905 910 Cys Gln Cys Gln Asn Lys Ala Asn Arg Thr Val Thr Asn Met Tyr Thr 915 920 925 Asn Val Ser Pro Gly Ser Ala Met Tyr Thr Asp Gly Phe His Ala Gln 930 935 940 Lys Ser Cys Gly Cys Lys Asn Asn Asp Met Tyr Gln Asn Gly Thr His 945 950 955 960 Pro His Lys Ser Cys Gly Cys Lys Asp Pro His Val Phe Ser Tyr His 965 970 975 Ile Asp Thr Gly Cys Val Asp Pro Glu Glu Asn Leu Gly Leu Trp Phe 980 985 990 Ala Leu Lys Ile Ala Gly Glu Asn Gly Val Ala Asn Ile Asp Asn Leu 995 1000 1005 Glu Ile Ile Glu Ala Gln Pro Leu Thr Gly Glu Ala Leu Ala Arg Val 1010 1015 1020 Lys Lys Arg G lu Gln Arg Trp Lys Gln Glu Arg Asp Lys Lys Arg Leu 1025 1030 1035 1040 Glu Thr Glu Lys Ala Val Gln Thr Ala Gln Gly Ala Ile Gln Asn Leu 1045 1050 1055 Phe Thr Asn Thr Gln Gln Asn Leu Leu Lys Phe Glu Thr Leu Phe Pro 1060 1065 1070 Gln Ile Val Asn Ala Glu Met Leu Val Gln Gln Ile Pro Tyr Val Tyr 1075 1080 1085 His Pro Phe Leu Ser Gly Ala Leu Pro Ala Val Ala Gly Met Asn Phe 1090 1095 1100 Lys Ile Val Gln Gln Leu Ser Ala Thr Ile Gly Asn Ala Arg Ser Leu 1105 1110 1115 1120 Tyr Asn Gln Arg Asn Leu Val Gln Asn Gly Thr Phe Ser Ser Gly Thr 1125 1130 1135 Gly Ser Trp His Val Ser Glu Gly Val Glu Val Gln Pro Leu Gln Asn 1140 1145 1150 Thr Ser Val Leu Val Leu Ser Glu Trp Ser His Glu Ala Ser Gln Gln 1155 1160 1165 Val Ser Val Asp Pro Asp Arg Gly Tyr Val Leu Arg Val Thr Ala Arg 1170 1175 1180 Lys Glu Gly Val Gly Lys Gly Thr Val Thr Leu Ser Asp Cys Ala Asp 1185 1190 1195 1200 Tyr Thr Glu Thr Leu Thr Phe Thr Ser Cys Asp Phe Asn Thr Ser Gly 1205 1210 1215 Ser Gln Thr Met Thr Ser Gly Thr Leu Ser Gly Phe Val Thr Lys Thr 1220 1225 1230 Leu Glu Ile Phe Pro Asp Thr Asp Arg Ile Arg Ile Asp Ile Gly Glu 1235 1240 1245 Thr Glu Gly Thr Phe Lys Ile Glu Ser Val Glu Leu Ile Cys Met Glu 1250 1255 1260 Gln Met Glu Asp Asp Leu 1265 1270

[0054] <210> 3 <211> 3933 <212> DNA <213> Bacillus thuringiensis <400> 3 tgaaagtacc atgaagctat ctttaatgtt agcatataca agcgaccgca aatgttacga 60 cacataagaa atacacgggg aggtttaaat atgaaacaaa attacaatga tgaatatgac 120 attatagata atggtggcag gggcgatcag tcaagatatc cccttacgaa tgcaccgagt 180 gctgaattac aagatatgaa ttatatagaa tggctggata attgtacact taaggagcag 240 gttgaactat tcagagatac ctcaactaca gttagggatg cactagctac aacggctggt 300 atcataaccg ctttactaag cgtttcaaat ccggcagctg cggccacagc aggaattatt 360 actatactta ttccacaact ttggccgagt ggttcagacg aagtaacttg ggaaaaattc 420 atggctgcag cagaaatctt aattcaaaaa caaattacag aggctgtacg taataaagca 480 cttacggaat tagagggtgt gtatagaact atacggcttt atcaactagc tgcagagcgt 540 tggaaccaaa ataagaatga tccacaggca caagaaagca taagaactca atttcgcgct 600 acgaatacga taatcgagtt cgcaatgccc tcatttagag tagcaggttt tgaagtgcca 660 ctgttaaatg tatatgcgga agctgccaac cttcaattag cattattacg agatgccgta 720 aaattcggga gagattgggg gctgccacag gatgaagttg acgatatata tagtgaacaa 780 ctact accaa gaatcgcaga acacacagac cattgtgtta cgtactttaa tagaggatta 840 gaggaagcaa aaaaattaaa agcaaattta aatgattatg ctcgttaccc ttgggcacag 900 tatataaatc actcgaagat acaaggaatt gagaattgga atctatttaa tgattatcga 960 agaaatatga caatactggt tttagatctt gttgcactat ggccaacata tgacccgaga 1020 cgatacagta tggtaacaaa atcagaacta acacgcgaat tgtatacaag tgtacgcggc 1080 gcattttacg gtcataataa tgattatgat cagaattttg aagaaattga gaggaatatt 1140 atctctccac cacacctagt cacttggcca ataaatttta cagtgtatac acaaaatgat 1200 tactattatc ctatggcggg tcttcaacat aagttcaatt acactgagtc tattgtttcc 1260 ctagagagtc cagtgacggg agtaacagga acctctaatc ttattaattt tgttacagct 1320 gatcctttca ttttagctgt aacaataatt ggttttggtc aattaggaac atctcttggt 1380 atatatgcaa tgtcttttgg gaggaagagc ggtagtatta gtcacgttgg ggatatagaa 1440 ataggaacgg atgattattt ggatatagtg gatcggattc ctgtgggcga ttccactcct 1500 aataaattat catggatgag cgctgctcct actactcttg gttcaacaac ctttttgcaa 1560 tatgtaagtt atgcctggag gcatcctagc gtagattcaa acaatagaat atcaattgat 1620 aagattaccc aaa ttccagc agtcaaggca ttttttatag atgacaatca tgtaaaagta 1680 attaaaggac ctggatctac aggaggagat ttagtcgctt tttctagaga gggttatggt 1740 cttagtatat cagtgtttat tcctggaagt gatttagtat cattttatag agtaaggatt 1800 cgttatgcga gtagtcagtc tgctaaagtc acaatgggtt tcggtggagg cgtagaggaa 1860 tcggagagca aatttaattt tcctgctacg tattctggtg gcaatctaac atataatagt 1920 tttgggtata taaatacttt ggcaattgca tctcaatcaa ctgcccaaac tattgaagtc 1980 tattttaggc aatacgagga agctgaattt attgtcgata aacttgaatt tattccgatg 2040 gaaatgtctt tggaagaata tcaagcggat caagacttag aaaaagcaag aaaggcagtg 2100 aacgccttgt ttacgagtga tgcgaagagt gaactgaaag taaatatcac agattacgcc 2160 gtagatcaag ctgccaattt agtagaatgt gtatcagagg acttccatgc acaagaaaaa 2220 aggatcctcc tggatcaagt caagtttgcg aaacgtctga gccaagcgcg gaacctatta 2280 aactacggag attttgagtc attagattgg tcgggagaga atggatggag aactagtcct 2340 catgtccatg tggcatctga taatccaatc tttaaagggc gctatctcca catgccaggt 2400 gcaatgagcc cacaatactc taacaatacg tatccaacgt acgcctatca aaaagtggat 2460 gagtcaaaat taaaatcct a tacacgttat ctcgtacgcg ggtttgtcgg taatagtaaa 2520 gacctagaat tactcgttga acgatacgga aaagaggtcc atgtcgagat ggatgtacca 2580 aatgatatcc gttctacctt accaatgaat gaatgcggag gctttgagcg gtgcggaaag 2640 gtatcatatc aagcggtatc ggatcacaca tgtacatgca aggatacagc tcgcatgaat 2700 acagattgcc agtgtaagga caaaataaac cacatgacaa caggcgtgta tacaagtatg 2760 ccagtaggta gtgccatgta tccggatggc taccatgcac acaaatcgtg taaatgtggt 2820 gacaaaacga tgtatgggaa cggaaaacat cctcataaat cctgtggatg caaagaccca 2880 catgtcttct cgtaccatat tgatacagga tgtgtggatc aagaagaaaa cctaggtttg 2940 tggttcgcct tgaaaattgc gagtgaaaat ggtgtcgcga acattgacaa tttagaaatc 3000 attgaagcac tgccgttaac tggagaggca ttagcacgtg tgaaaaaacg agaacacaaa 3060 tggaaacaag aaatggaaca gaaacgttgt aaaacagaag aagcggtaca ggctgcgcaa 3120 acagcgatta acaccctatt cacaaataca caatacaatc gcttgaaatt tgagacatta 3180 ttcccgcaca ttctgcatgc tgatgaactt gtacaacgta ttccatatgt gtatcaccca 3240 ttcttacttg gcgcatatcc tgatgtacca ggaatgaact atgatatctt ccagcaactc 3300 tctgctttgg tgaatcaagc acgt ggatta tatgacatgc gaaatctagt acgaaatggc 3360 acattcagtg ccggcatagg aaattggcag gtaacagacg gagtagcgac acaaccagaa 3420 ggaaatacct ctgtactgat tctacgagaa tggagcgata aggccatcca acacctacga 3480 atccatgcag aacgcgggta tgtgttacgt gtaacagcac gaaaagaagg taacggtgac 3540 ggctatgtcg tgattcatga ttgtgataat cagcaggaaa aattaacatt tacagcgtgt 3600 gattatacaa cgatggggtc aagtacgggt acacaaacga tgatgacatc gcctacaaat 3660 tgcatgccat gtaactccac aacatggaag gaagaaatga aagcacttgt acctatgtta 3720 agtggatatg tgacaaaaac agcggaaatc ttcccagata ccgatcgtat tcacattgaa 3780 atcggcgaaa cagaaggcac atttaaaatc gaaagtgtgg aactcatctg tatggaacac 3840 atggaagagc atgcatatga tatggaaggt gatatagaag caaatatacc accaattgta 3900 cgaccaccga tcatgcc

<210> 4 <211> 1280 <212> PRT <213> Bacillus thuringiensis <400> 4 Met Lys Gln Asn Tyr Asn Asp Glu Tyr Asp Ile Ile Asp Asn Gly Gly 1 5 10 15 Arg Gly Asp Gln Ser Arg Tyr Pro Leu Thr Asn Ala Pro Ser Ala Glu 20 25 30 Leu Gln Asp Met Asn Tyr Ile Glu Trp Leu Asp Asn Cys Thr Leu Lys 35 40 45 Glu Gln Val Glu Leu Phe Arg Asp Thr Ser Thr Thr Val Arg Asp Ala 50 55 60 Leu Ala Thr Thr Ala Gly Ile Ile Thr Ala Leu Leu Ser Val Ser Asn 65 70 75 80 Pro Ala Ala Ala Ala Ala Thr Ala Gly Ile Ile Thr Ile Leu Ile Pro Gln 85 90 95 Leu Trp Pro Ser Gly Ser Asp Glu Val Thr Trp Glu Lys Phe Met Ala 100 105 110 Ala Ala Glu Ile Leu Ile Gln Lys Gln Ile Thr Glu Ala Val Arg Asn 115 120 125 Lys Ala Leu Thr Glu Leu Glu Gly Val Tyr Arg Thr Ile Arg Leu Tyr 130 135 140 Gln Leu Ala Ala Glu Arg Trp Asn Gln Asn Lys Asn Asp Pro Gln Ala 145 150 155 160 Gln Glu Ser Ile Arg Thr Gln Phe Arg Ala Thr Asn Thr Ile Ile Glu 165 170 175 Phe Ala Met Pro Ser Phe Arg Val Ala Gly Phe Glu Val Pro Leu Leu 180 185 190 Asn Val Tyr A la Glu Ala Ala Asn Leu Gln Leu Ala Leu Leu Arg Asp 195 200 205 Ala Val Lys Phe Gly Arg Asp Trp Gly Leu Pro Gln Asp Glu Val Asp 210 215 220 Asp Ile Tyr Ser Glu Gln Leu Leu Pro Arg Ile Ala Glu His Thr Asp 225 230 235 240 His Cys Val Thr Tyr Phe Asn Arg Gly Leu Glu Glu Ala Lys Lys Leu 245 250 255 Lys Ala Asn Leu Asn Asp Tyr Ala Arg Tyr Pro Trp Ala Gln Tyr Ile 260 265 270 270 Asn His Ser Lys Ile Gln Gly Ile Glu Asn Trp Asn Leu Phe Asn Asp 275 280 285 Tyr Arg Arg Asn Met Thr Ile Leu Val Leu Asp Leu Val Ala Leu Trp 290 295 300 Pro Thr Tyr Asp Pro Arg Arg Tyr Ser Met Val Thr Lys Ser Glu Leu 305 310 315 320 Thr Arg Glu Leu Tyr Thr Ser Val Arg Gly Ala Phe Tyr Gly His Asn 325 330 335 Asn Asp Tyr Asp Gln Asn Phe Glu Glu Ile Glu Arg Asn Ile Ile Ser 340 345 350 Pro Pro His Leu Val Thr Trp Pro Ile Asn Phe Thr Val Tyr Thr Gln 355 360 365 Asn Asp Tyr Tyr Tyr Pro Met Ala Gly Leu Gln His Lys Phe Asn Tyr 370 375 380 Thr Glu Ser Ile Val Ser Leu Glu Ser Pro Val Thr Gly Val Thr Gly 385 390 395 400 400 Thr Ser AsnLeu Ile Asn Phe Val Thr Ala Asp Pro Phe Ile Leu Ala 405 410 415 Val Thr Ile Ile Gly Phe Gly Gln Leu Gly Thr Ser Leu Gly Ile Tyr 420 425 430 Ala Met Ser Phe Gly Arg Lys Ser Gly Ser Ile Ser His Val Gly Asp 435 440 445 Ile Glu Ile Gly Thr Asp Asp Asp Tyr Leu Asp Ile Val Asp Arg Ile Pro 450 455 460 Val Gly Asp Ser Thr Pro Asn Lys Leu Ser Trp Met Ser Ala Ala Pro 465 470 475 480 480 Thr Thr Leu Gly Ser Thr Thr Phe Leu Gln Tyr Val Ser Tyr Ala Trp 485 490 495 Arg His Pro Ser Val Asp Ser Asn Asn Arg Ile Ser Ile Asp Lys Ile 500 505 510 Thr Gln Ile Pro Ala Val Lys Ala Phe Phe Ile Asp Asp Asn His Val 515 520 525 Lys Val Ile Lys Gly Pro Gly Ser Thr Gly Gly Asp Leu Val Ala Phe 530 535 540 Ser Arg Glu Gly Tyr Gly Leu Ser Ile Ser Val Phe Ile Pro Gly Ser 545 550 555 560 Asp Leu Val Ser Phe Tyr Arg Val Arg Ile Arg Tyr Ala Ser Ser Gln 565 570 575 Ser Ala Lys Val Thr Met Gly Phe Gly Gly Gly Val Glu Glu Ser Glu 580 585 590 Ser Lys Phe Asn Phe Pro Ala Thr Tyr Ser Gly Gly Asn Leu Thr Tyr 595 600 605 Asn Ser Phe Gly Tyr Ile Asn Thr Leu Ala Ile Ala Ser Gln Ser Thr 610 615 620 Ala Gln Thr Ile Glu Val Tyr Phe Arg Gln Tyr Glu Glu Ala Glu Phe 625 630 635 640 640 Ile Val Asp Lys Leu Glu Phe Ile Pro Met Glu Met Ser Leu Glu Glu 645 650 655 Tyr Gln Ala Asp Gln Asp Leu Glu Lys Ala Arg Lys Ala Val Asn Ala 660 665 670 Leu Phe Thr Ser Asp Ala Lys Ser Glu Leu Lys Val Asn Ile Thr Asp 675 680 685 Tyr Ala Val Asp Gln Ala Ala Asn Leu Val Glu Cys Val Ser Glu Asp 690 695 700 Phe His Ala Gln Glu Lys Arg Ile Leu Leu Asp Gln Val Lys Phe Ala 705 710 715 715 720 Lys Arg Leu Ser Gln Ala Arg Asn Leu Leu Asn Tyr Gly Asp Phe Glu 725 730 735 Ser Leu Asp Trp Ser Gly Glu Asn Gly Trp Arg Thr Ser Pro His Val 740 745 750 His Val Ala Ser Asp Asn Pro Ile Phe Lys Gly Arg Tyr Leu His Met 755 760 765 Pro Gly Ala Met Ser Pro Gln Tyr Ser Asn Asn Thr Tyr Pro Thr Tyr 770 775 780 Ala Tyr Gln Lys Val Asp Glu Ser Lys Leu Lys Ser Tyr Thr Arg Tyr 785 790 795 800 Leu Val Arg Gly Phe Val Gly Asn Ser Lys Asp Leu Glu Leu Leu Val 805 810 815 Glu Arg Tyr Gl y Lys Glu Val His Val Glu Met Asp Val Pro Asn Asp 820 825 830 Ile Arg Ser Thr Leu Pro Met Asn Glu Cys Gly Gly Phe Glu Arg Cys 835 840 845 Gly Lys Val Ser Tyr Gln Ala Val Ser Asp His Thr Cys Thr Cys Lys 850 855 860 Asp Thr Ala Arg Met Asn Thr Asp Cys Gln Cys Lys Asp Lys Ile Asn 865 870 875 880 His Met Thr Thr Gly Val Tyr Thr Ser Met Pro Val Gly Ser Ala Met 885 890 895 Tyr Pro Asp Gly Tyr His Ala His Lys Ser Cys Lys Cys Gly Asp Lys 900 905 910 910 Thr Met Tyr Gly Asn Gly Lys His Pro His Lys Ser Cys Gly Cys Lys 915 920 925 Asp Pro His Val Phe Ser Tyr His Ile Asp Thr Gly Cys Val Asp Gln 930 935 940 Glu Glu Asn Leu Gly Leu Trp Phe Ala Leu Lys Ile Ala Ser Glu Asn 945 950 955 960 Gly Val Ala Asn Ile Asp Asn Leu Glu Ile Ile Glu Ala Leu Pro Leu 965 970 975 Thr Gly Glu Ala Leu Ala Arg Val Lys Lys Arg Glu His Lys Trp Lys 980 985 990 Gln Glu Met Glu Gln Lys Arg Cys Lys Thr Glu Glu Ala Val Gln Ala 995 1000 1005 Ala Gln Thr Ala Ile Asn Thr Leu Phe Thr Asn Thr Gln Tyr Asn Arg 1010 1015 1020 Leu Lys Phe G lu Thr Leu Phe Pro His Ile Leu His Ala Asp Glu Leu 1025 1030 1035 1040 Val Gln Arg Ile Pro Tyr Val Tyr His Pro Phe Leu Leu Gly Ala Tyr 1045 1050 1055 Pro Asp Val Pro Gly Met Asn Tyr Asp Ile Phe Gln Gln Leu Ser Ala 1060 1065 1070 Leu Val Asn Gln Ala Arg Gly Leu Tyr Asp Met Arg Asn Leu Val Arg 1075 1080 1085 Asn Gly Thr Phe Ser Ala Gly Ile Gly Asn Trp Gln Val Thr Asp Gly 1090 1095 1100 Val Ala Thr Gln Pro Glu Gly Asn Thr Ser Val Leu Ile Leu Arg Glu 1105 1110 1115 1120 Trp Ser Asp Lys Ala Ile Gln His Leu Arg Ile His Ala Glu Arg Gly 1125 1130 1135 Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly Asn Gly Asp Gly Tyr 1140 1145 1150 Val Val Ile His Asp Cys Asp Asn Gln Gln Glu Lys Leu Thr Phe Thr 1155 1160 1165 Ala Cys Asp Tyr Thr Thr Met Gly Ser Ser Thr Gly Thr Gln Thr Met 1170 1175 1180 Met Thr Ser Pro Thr Asn Cys Met Pro Cys Asn Ser Thr Thr Trp Lys 1185 1190 1195 1200 Glu Glu Met Lys Ala Leu Val Pro Met Leu Ser Gly Tyr Val Thr Lys 1205 1210 1215 Thr Ala Glu Ile Phe Pro Asp Thr Asp Arg Ile His I le Glu Ile Gly 1220 1225 1230 Glu Thr Glu Gly Thr Phe Lys Ile Glu Ser Val Glu Leu Ile Cys Met 1235 1240 1245 Glu His Met Glu Glu His Ala Tyr Asp Met Glu Gly Asp Ile Glu Ala 1250 1255 1260 Asn Ile Pro Pro Ile Val Arg Pro Pro Ile Met Pro Pro Thr Asn Val 1265 1270 1275 1280

[0056] <210> 5 <211> 4332 <212> DNA <213> Bacillus thuringiensis <400> 5 taggagtgct tcgagtaggt gaatacaaac atcccatttt gttgtgtagt caagatcccc 60 catggctttg cgtatgtacc gaatgaaaca catataattg catacaacct tgattgtcta 120 tctgtcatag aagaaaagcg cagaaaaacc atccaagtgg atggatgcgg tcctgttgaa 180 gtcacactca atcttttaaa agtggtggga tgtattcctt atatagtgaa tgcaacgata 240 gaagctgaat gtggtagtaa ctgtgattgc gattcgcatg gaaaatatca cattagcgtt 300 tgytgttcgg gaagtatttg cctagataat gtgctgaaat gtggtgtcga atgtttacca 360 cattacgaga tggattgtca caatgttatt gtttcgaatt taaaggtaaa gccactgcaa 420 gattacggca ttgaaatatt gcagtttaca gggacagtgg aatttaagca cattccttat 480 gaataaaaca tctatctttg ttcaaagtgt atcaaaatgt gaacatattt gaaagaacat 540 aaaaaaatag aaaaaggagg cttatatatg aattcaaatt ataacaatga acatgagatc 600 ttagatatga ataaccgaag ttatcaaacc agatatcccc ttgcgaatgc accaggtgtt 660 gaattacaac aaatgagtta taaggattgg atggatagat gtgaacaaga gtcgctggca 720 atcacattta aatccgttat tacaactgct ttagatatta cgtctgcaat cctcggtgcg 780 gcaaaa tctc caaaagctaa agtagcaaga gctgcagttc aagtccttaa tactgttatt 840 aaattgcttt ggcccgatcc agaaaaacct tctgaacccg catacgatat agatttcatc 900 tggaaagagc tgatagggag agtcgaaata ctaattgaag aaaaaattga ccgagaagct 960 tataacgccg caattggaag gttatcagga ttaaagagag ctttaagttt atatcaagaa 1020 tcgtttgaga cttggatcga cgatgaaaat gaccctgagt tacaggaaga tgtacgaatg 1080 cgctttacat ctacactgtt tgaacttgtg actacaattg aaacatttaa atacagtgga 1140 caagagctaa atttattgac agtttttgta caagctgcag attttcattt aatgttatta 1200 caacaaggag taatgtatgg agttcggtgg ggctttgatc agagaacggt ggattctttt 1260 tatcaaaatg acagaggaga aggcttaaaa aatttgctac caaagtattc tgattattgc 1320 acttattggt atgatcaagg tttgaataga gcgaaaaact tgaaggcaaa tttatcagat 1380 acagtaaggt atccttgggc tgcaaactta gaaaatatga gtgtgttaca agagttagaa 1440 gattggaacc tttataatga ttatcgaaga gacatgacaa tcttggtatt agatttggtt 1500 gctgtatggc caacatatga tctccattat tacgataatg gaaactatgg ggtacagtcg 1560 gaactgacac gatctatata ctctcaagca gtaggaaatg taatgggaac tgtatttaca 1620 aaagagcaat acg aggttag tttcgttcgc ccgccacact tagttacttg gttagaaaaa 1680 atgtttattc aaataagacc aacagagcaa ggcgcaccca tcgatgcgac aatggctggt 1740 gtcagtctat attattctta ttcaggttgg gataatacag ttgatgacat acttctagga 1800 tatccaacat attcgagtag tgaaattcgt gtgctttcaa aaagtaaagt aatcgttcaa 1860 gatcaagaaa aaaatcgagc gatttacaat acagacctcc aacatgataa actagtagat 1920 cgatttgttt tttatcaaaa tagtggggaa gttaattatg ctggtagaga taatccttca 1980 agctataaaa cattcgcatg ggatacggat attaccaatt atagtagtca aatgacatgg 2040 ataaatggcc cagtaaatga agggcatttt ggttatattc aggcttatgc accagaatgg 2100 atccctgcaa gttgtgaacc atttaataat atagtagatg cagaagatgt gattactcaa 2160 ataccggcag tgaaggctcg agaattaaga tatggtgcac gtgttataaa aggggtgggt 2220 aatacaggtg gagatttagt gtctattgca ccacatggtt tgtgtgagtt gtacgtgtca 2280 tttccaaatg tagtccgaag atatcaggtt cggatacatt atgcatgtca ggatccgacc 2340 aaaataaatc taaatatagg ggattcacgt catgatatta cgcttccatc tacgtattct 2400 ggaggagcat tgacatacga ttcatttggt tatgcaacaa gtgaatacag ctatctgttt 2460 tacgctgatt tttatgacg a gaaacagata gtacgtttgg gaaattcttt tgatatatca 2520 cagcaagatg tcatcattga taagattgaa tttattcctg ttgacatctt ctatgcagag 2580 gaacaagatt tagaaaaagc aaggaaggca gtgaacgctc tgttcacgaa tgacgcaaag 2640 aatgccttac aactggatgt cacagattac gcggtagacc aagctgccaa tctggtagag 2700 tgtgtatcgg atgaattcca tgcccaagaa aaaatgatcc tactggatca agtcaagttt 2760 gcgaaacgtc taagtcaagc gcgaaaccta ttaaattatg gagattttga atcctcagat 2820 tggtcgggag agaatggatg gagaaccagt cctcatgtcc atgtggcatc tgataatcca 2880 atctttaagg ggcgctatct ccacatgtca ggtgcgatga gccctcaatt ctctaacaat 2940 atttatccaa cgtatgcgta tcaaaaagtg gatgagtcca aattaaaatc ctatacacgt 3000 tatctcgtac gtggattcgt agggaatagt aaagacctag aattattggt agaacgatat 3060 ggaaaagatg tatatgtaga aatggatgtc ccaaatgata tccggtatac gttaccaaga 3120 aatgaatgtg gtgactttga tcgatgcaaa cctgcctcgt atcaaacgaa acctcctcat 3180 acctgcacat gtaaagatcc cgctgtagct catacggatt gtcagtgcaa agataaggga 3240 aatcatactt cgacgaacat gtatacaaat ggaccaacag gtagtgaagt gtctacgaat 3300 ggattccatg cccacaaatc ttgt gggtgc ggtgacaaac acatggataa gagtggaaca 3360 catccgcaca aatcctgtgg atgcaaagac ccacatgttt tcacgtacca tattgacaca 3420 gggtgtgtcg acacggaaga aaacttaggt ctattctttg ctttaaaaat tgcgagcgaa 3480 aatggtgtcg caaacattga taatttagaa atcattgaag cacagccgct aacaggagaa 3540 gcattagcac gtgtgaaaaa acgagaacaa agatggaaac aagaaatgac aaagaaacgg 3600 ttagaaacag agacagctgt acaagcagcg cawgatgcga ttcagaatct atttacaaac 3660 acgcagtaca atcgtctgaa atttgaaacg ctgttccctc acattgtcaa tgcggagttg 3720 ttggtacaac agattccgta tgtataccac ccattcttga gcggggcact gccagtcgta 3780 ccaggcatga atttcgatat ttttcaacaa ctttctttct tgattgatac agcaaagagg 3840 ttatatgagg cgcgaaatct cgtgcaaaat ggcacattca gttccggaac aggaagctgg 3900 tacgtgacag ctggagtgga agtgcaccga cttcaaaata catctgtgct tgtcctatct 3960 gaatggagtc atgaagcgtc ccagcaggta cggattgatt cggatcgcgg atatgtgtta 4020 cgtgtaacag cacgaaaaga aggcgcagga aaaggtactg tgacactaag tgattgtgca 4080 gactatacag aaacactgtc ctttacatct tgtgactata atacggttgg tacccaaacg 4140 atgactaaag gtacgttatc gggatttgt g accaaaacgt tggaaaattt tccagacaca 4200 gatcgtattc gcattgatat tggggaaacc gaaggtacgt ttaaagtaga aagtgtagaa 4260 ctgatttgta tggaacaaat ggaggaccat ttatatgata tggcgggaaa cttagaacag 4320 gatctaacat ag 4332

<210> 6 <211> 1253 <212> PRT <213> Bacillus thuringiensis <400> 6 Met Asn Ser Asn Tyr Asn Asn Glu His Glu Ile Leu Asp Met Asn Asn 1 5 10 15 Arg Ser Tyr Gln Thr Arg Tyr Pro Leu Ala Asn Ala Pro Gly Val Glu 20 25 30 Leu Gln Gln Met Ser Tyr Lys Asp Trp Met Asp Arg Cys Glu Gln Glu 35 40 45 Ser Leu Ala Ile Thr Phe Lys Ser Val Ile Thr Thr Ala Leu Asp Ile 50 55 60 Thr Ser Ala Ile Leu Gly Ala Ala Lys Ser Pro Lys Ala Lys Val Ala 65 70 75 80 Arg Ala Ala Val Gln Val Leu Asn Thr Val Ile Lys Leu Leu Trp Pro 85 90 95 Asp Pro Glu Lys Pro Ser Glu Pro Ala Tyr Asp Ile Asp Phe Ile Trp 100 105 110 Lys Glu Leu Ile Gly Arg Val Glu Ile Leu Ile Glu Glu Lys Ile Asp 115 120 125 Arg Glu Ala Tyr Asn Ala Ala Ile Gly Arg Leu Ser Gly Leu Lys Arg 130 135 140 Ala Leu Ser Leu Tyr Gln Glu Ser Phe Glu Thr Trp Ile Asp Asp Glu 145 150 155 160 Asn Asp Pro Glu Leu Gln Glu Asp Val Arg Met Arg Phe Thr Ser Thr 165 170 175 Leu Phe Glu Leu Val Thr Thr Ile Glu Thr Phe Lys Tyr Ser Gly Gln 180 185 190 Glu Leu Asn L eu Leu Thr Val Phe Val Gln Ala Ala Asp Phe His Leu 195 200 205 Met Leu Leu Gln Gln Gly Val Met Tyr Gly Val Arg Trp Gly Phe Asp 210 215 220 Gln Arg Thr Val Asp Ser Phe Tyr Gln Asn Asp Arg Gly Glu Gly Leu 225 230 235 240 Lys Asn Leu Leu Pro Lys Tyr Ser Asp Tyr Cys Thr Tyr Trp Tyr Asp 245 250 255 Gln Gly Leu Asn Arg Ala Lys Asn Leu Lys Ala Asn Leu Ser Asp Thr 260 265 270 270 Val Arg Tyr Pro Trp Ala Ala Asn Leu Glu Asn Met Ser Val Leu Gln 275 280 285 Glu Leu Glu Asp Trp Asn Leu Tyr Asn Asp Tyr Arg Arg Asp Met Thr 290 295 300 Ile Leu Val Leu Asp Leu Val Ala Val Trp Pro Thr Tyr Asp Leu His 305 310 315 320 Tyr Tyr Asp Asn Gly Asn Tyr Gly Val Gln Ser Glu Leu Thr Arg Ser 325 330 335 Ile Tyr Ser Gln Ala Val Gly Asn Val Met Gly Thr Val Phe Thr Lys 340 345 350 Glu Gln Tyr Glu Val Ser Phe Val Arg Pro Pro His Leu Val Thr Trp 355 360 365 Leu Glu Lys Met Phe Ile Gln Ile Arg Pro Thr Glu Gln Gly Ala Pro 370 375 380 Ile Asp Ala Thr Met Ala Gly Val Ser Leu Tyr Tyr Ser Tyr Ser Gly 385 390 395 400 Trp Asp AsnThr Val Asp Asp Ile Leu Leu Gly Tyr Pro Thr Tyr Ser 405 410 415 Ser Ser Glu Ile Arg Val Leu Ser Lys Ser Lys Val Ile Val Gln Asp 420 425 430 Gln Glu Lys Asn Arg Ala Ile Tyr Asn Thr Asp Leu Gln His Asp Lys 435 440 445 Leu Val Asp Arg Phe Val Phe Tyr Gln Asn Ser Gly Glu Val Asn Tyr 450 455 460 Ala Gly Arg Asp Asn Pro Ser Ser Tyr Lys Thr Phe Ala Trp Asp Thr 465 470 475 480 Asp Ile Thr Asn Tyr Ser Ser Gln Met Thr Trp Ile Asn Gly Pro Val 485 490 495 Asn Glu Gly His Phe Gly Tyr Ile Gln Ala Tyr Ala Pro Glu Trp Ile 500 505 510 Pro Ala Ser Cys Glu Pro Phe Asn Asn Ile Val Asp Ala Glu Asp Val 515 520 525 Ile Thr Gln Ile Pro Ala Val Lys Ala Arg Glu Leu Arg Tyr Gly Ala 530 535 540 Arg Val Ile Lys Gly Val Gly Asn Thr Gly Gly Asp Leu Val Ser Ile 545 550 555 560 Ala Pro His Gly Leu Cys Glu Leu Tyr Val Ser Phe Pro Asn Val Val 565 570 575 Arg Arg Tyr Gln Val Arg Ile His Tyr Ala Cys Gln Asp Pro Thr Lys 580 585 590 Ile Asn Leu Asn Ile Gly Asp Ser Arg His Asp Ile Thr Leu Pro Ser 595 600 605 Thr Tyr Ser Gly Gly Ala Leu Thr Tyr Asp Ser Phe Gly Tyr Ala Thr 610 615 620 Ser Glu Tyr Ser Tyr Leu Phe Tyr Ala Asp Phe Tyr Asp Glu Lys Gln 625 630 635 635 640 Ile Val Arg Leu Gly Asn Ser Phe Asp Ile Ser Gln Gln Asp Val Ile 645 650 655 Ile Asp Lys Ile Glu Phe Ile Pro Val Asp Ile Phe Tyr Ala Glu Glu 660 665 670 Gln Asp Leu Glu Lys Ala Arg Lys Ala Val Asn Ala Leu Phe Thr Asn 675 680 685 Asp Ala Lys Asn Ala Leu Gln Leu Asp Val Thr Asp Tyr Ala Val Asp 690 695 700 Gln Ala Ala Asn Leu Val Glu Cys Val Ser Asp Glu Phe His Ala Gln 705 710 715 720 Glu Lys Met Ile Leu Leu Asp Gln Val Lys Phe Ala Lys Arg Leu Ser 725 730 735 Gln Ala Arg Asn Leu Leu Asn Tyr Gly Asp Phe Glu Ser Ser Asp Trp 740 745 750 Ser Gly Glu Asn Gly Trp Arg Thr Ser Pro His Val His Val Ala Ser 755 760 765 Asp Asn Pro Ile Phe Lys Gly Arg Tyr Leu His Met Ser Gly Ala Met 770 775 780 Ser Pro Gln Phe Ser Asn Asn Ile Tyr Pro Thr Tyr Ala Tyr Gln Lys 785 790 795 800 Val Asp Glu Ser Lys Leu Lys Ser Tyr Thr Arg Tyr Leu Val Arg Gly 805 810 815 Phe Val Gly As n Ser Lys Asp Leu Glu Leu Leu Val Glu Arg Tyr Gly 820 825 830 Lys Asp Val Tyr Val Glu Met Asp Val Pro Asn Asp Ile Arg Tyr Thr 835 840 845 Leu Pro Arg Asn Glu Cys Gly Asp Phe Asp Arg Cys Lys Pro Ala Ser 850 855 860 Tyr Gln Thr Lys Pro Pro His Thr Cys Thr Cys Lys Asp Pro Ala Val 865 870 875 880 Ala His Thr Asp Cys Gln Cys Lys Asp Lys Gly Asn His Thr Ser Thr 885 890 895 895 Asn Met Tyr Thr Asn Gly Pro Thr Gly Ser Glu Val Ser Thr Asn Gly 900 905 910 Phe His Ala His Lys Ser Cys Gly Cys Gly Asp Lys His Met Asp Lys 915 920 925 Ser Gly Thr His Pro His Lys Ser Cys Gly Cys Lys Asp Pro His Val 930 935 940 Phe Thr Tyr His Ile Asp Thr Gly Cys Val Asp Thr Glu Glu Asn Leu 945 950 955 960 Gly Leu Phe Phe Ala Leu Lys Ile Ala Ser Glu Asn Gly Val Ala Asn 965 970 975 Ile Asp Asn Leu Glu Ile Ile Glu Ala Gln Pro Leu Thr Gly Glu Ala 980 985 990 Leu Ala Arg Val Lys Lys Arg Glu Gln Arg Trp Lys Gln Glu Met Thr 995 1000 1005 Lys Lys Arg Leu Glu Thr Glu Thr Ala Val Gln Ala Ala Asp Ala Ile 1010 1015 1020 Gln Asn Leu Phe Thr Asn Thr Gln Tyr Asn Arg Leu Lys Phe Glu Thr 1025 1030 1035 1040 Leu Phe Pro His Ile Val Asn Ala Glu Leu Leu Val Gln Gln Ile Pro 1045 1050 1055 Tyr Val Tyr His Pro Phe Leu Ser Gly Ala Leu Pro Val Val Pro Gly 1060 1065 1070 Met Asn Phe Asp Ile Phe Gln Gln Leu Ser Phe Leu Ile Asp Thr Ala 1075 1080 1085 Lys Arg Leu Tyr Glu Ala Arg Asn Leu Val Gln Asn Gly Thr Phe Ser 1090 1095 1100 Ser Gly Thr Gly Ser Trp Tyr Val Thr Ala Gly Val Glu Val His Arg 1105 1110 1115 1120 Leu Gln Asn Thr Ser Val Leu Val Leu Ser Glu Trp Ser His Glu Ala 1125 1130 1135 Ser Gln Gln Val Arg Ile Asp Ser Asp Arg Gly Tyr Val Leu Arg Val 1140 1145 1150 Thr Ala Arg Lys Glu Gly Ala Gly Lys Gly Thr Val Thr Leu Ser Asp 1155 1160 1165 Cys Ala Asp Tyr Thr Glu Thr Leu Ser Phe Thr Ser Cys Asp Tyr Asn 1170 1175 1180 Thr Val Gly Thr Gln Thr Met Thr Lys Gly Thr Leu Ser Gly Phe Val 1185 1190 1195 1200 Thr Lys Thr Leu Glu Asn Phe Pro Asp Thr Asp Arg Ile Arg Ile Asp 1205 1210 1215 Ile Gly Glu Thr Glu Gly Thr Phe Lys Val Glu Ser Val Glu Leu Ile 1220 1225 1230 Cys Met Glu Gln Met Glu Asp His Leu Tyr Asp Met Ala Gly Asn Leu 1235 1240 1245 Glu Gln Asp Leu Thr 1250

[0058] <210> 7 <211> 1548 <212> DNA <213> Bacillus thuringiensis <400> 7 aggagagttt gatcctggct caggatgaac gctggcggcg tgcctaatac atgcaagtcg 60 agcgaatgga ttgagagctt gctctcaaga agttagcggc ggacgggtga gtaacacgtg 120 ggtaacctgc ccataagact gggataactc cgggaaaccg gggctaatac cggataatat 180 tttgaactgc atggttcgaa attgaaaggc ggcttcggct gtcacttatg gatggacccg 240 cgtcgcatta gctagttggt gaggtaacgg ctcaccaagg caacgatgcg tagccgacct 300 gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 360 gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg agtgatgaag 420 gctttcgggt cgtaaaactc tgttgttagg gaagaacaag tgctagttga ataagctggc 480 accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540 cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg tggtttctta 600 agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg ggagacttga 660 gtgcagaaga ggaaagtgga attccatgtg tagcggtgaa atgcgtagag atatggagga 720 acaccagtgg cgaaggcgac tttctggtct gtaactgaca ctgaggcgcg aaagcgtggg 780 gagcaa acag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840 gagggtttcc gccctttagt gctgaagtta acgcattaag cactccgcct ggggagtacg 900 gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960 tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga aaaccctaga 1020 gatagggctt ctccttcggg agcagagtga caggtggtgc atggttgtcg tcagctcgtg 1080 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag ttgccatcat 1140 taagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1200 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggacg gtacaaagag 1260 ctgcaagacc gcgaggtgga gctaatctca taaaaccgtt ctcagttcgg attgtaggct 1320 gcaactcgcc tacatgaagc tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1380 tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta acacccgaag 1440 tcggtggggt aacctttatg gaggcaagcc gcctaaggtg ggacaagatg attggggtga 1500 agtcgtaaca aggtagcgta tcggaaggtg cggctggatc acctcctt 1548

<210> 8 <211> 23 <212> DNA <213> Artificial Sequence "n" in the sequence represents inosine <400> 8 taygyncargy ngcnaayyt hma 23

<210> 9 <211> 17 <212> DNA <213> Artificial Sequence <400> 9 atcycchccy gtawrhc 17

<210> 10 <211> 23 <212> DNA <213> Artificial Sequence <400> 10 cactgcaaga tcatggctgt cac 23

<210> 11 <211> 25 <212> DNA <213> Artificial Sequence <400> 11 aattagcagg cttagcacag actta 25

<210> 12 <211> 29 <212> DNA <213> Artificial Sequence <400> 12 tcttgactga tcgcccctgc caccattat 29

<210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 tcggtggagg cgtagaggaa tcggagagca 30

<210> 14 <211> 48 <212> DNA <213> Artificial Sequence <400> 14 ccatgggtat gaaacaaaat tacaatgatg aatatgacat tatagata 48

<210> 15 <211> 32 <212> DNA <213> Artificial Sequence <400> 15 ccatggttac attggtaggt ggcatgatcg gt 32

<210> 16 <211> 32 <212> DNA <213> Artificial Sequence <400> 16 ggatggtttt tctgcgcttt tcttctatga ca 32

<210> 17 <211> 36 <212> DNA <213> Artificial Sequence <400> 17 aagtatttgc ctagataatg tgctgaaatg tggtgt 36

<210> 18 <211> 46 <212> DNA <213> Artificial Sequence <400> 18 gcatgcagat gaattcaaat tataacaatg aacatgagat cttaga 46

<210> 19 <211> 35 <212> DNA <213> Artificial Sequence <400> 19 gcatgcttgt tagatcctgt tctaagtttc ccgcc 35

<210> 20 <211> 44 <212> DNA <213> Artificial Sequence <400> 20 gcatgcagat gaaccaaaat gacaacaaga atgaatatga aata 44

<210> 21 <211> 39 <212> DNA <213> Artificial Sequence <400> 21 gcatgcttaa gtcgtcttcc atctgttcca tacaaatga 39

[Brief description of the drawings]

FIG. 1 shows the general structure of the Cry protein. C
There are five regions (blocks 1 to 5) containing amino acid sequences that are relatively conserved in the ry protein.

FIG. 2. Cry protein (CryE6) in E. coli.
L, expression of CryE6S, and CryE6Q)
DS-PAGE (top) and Western blotting (bottom). Lane 3: purified CryE6L, CryE6S, or CryE6Q (protein amount 1 μg). Lanes 1 and 2: plasmid containing the gene of the present invention (pQ
(E70-cry6L, pQE60-cryE6S, or pQE70-cry6Q) in the case of a liquid (10 μg protein) in which cells were separated from the culture of Escherichia coli transformed with E. coli and crushed. Lane 1 is IPT
Culture was performed with G added. Lane 2 was cultured without adding IPTG. Lane 4: cells isolated from a culture of a culture containing IPTG of Escherichia coli transformed with a parent plasmid (pQE70 or pQE60 / M15 [pREP4]) not containing the gene of the present invention, and disrupted cells (protein content 10 μg).

Claims (10)

[Claims] 1. Bacillus thuringiensis TK-E6 strain 2. A gene (cryE6L) containing the nucleotide sequence at positions 161 to 9773 of SEQ ID NO: 1. 3. A gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 2. 4. A protein comprising the amino acid sequence of SEQ ID NO: 2 (CryE6L). 5. A gene (cryE6S) comprising the nucleotide sequence at positions 91 to 9333 of SEQ ID NO: 3. 6. A gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 4. 7. A protein comprising the amino acid sequence of SEQ ID NO: 4 (CryE6S). 8. A gene (cryE6Q) comprising the nucleotide sequence at positions 568 to 4332 of SEQ ID NO: 5. 9. A gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 6. 10. A protein comprising the amino acid sequence of SEQ ID NO: 6 (CryE6Q).