阅读说明：本技术 一种抗虫蛋白Cry1ABn39、编码基因和应用 (Insect-resistant protein Cry1ABn39, encoding gene and application ) 是由 舍亚涛 王长海 王爱华 侯兴松 刘野 姜辉 权永刚 王晓乐 高伟德 王博涵 于 2020-06-16 设计创作，主要内容包括：本发明提出了一种抗虫蛋白Cry1ABn39编码基因和其在转基因抗虫玉米育种领域的应用。该抗虫蛋白Cry1ABn39的氨基酸序列如SEQ ID No.1所示,编码基因的碱基序列如SEQ ID No.2所示。所述应用是编码基因在培育抗虫转基因玉米植株中的应用,在制备重组抗虫蛋白Cry1ABn39中的应用,以及抗虫蛋白Cry1ABn39在制备抗虫剂中的应用。本发明通过对抗虫蛋白的人工改造及其植物表达载体的优化增加了其在转基因材料中的表达量且不影响其原本抗虫功能和转基因玉米的其他农艺性状,因此能够更容易获得抗虫效果。(The invention provides an insect-resistant protein Cry1ABn39 encoding gene and application thereof in the field of transgenic insect-resistant corn breeding. The amino acid sequence of the insect-resistant protein Cry1ABn39 is shown as SEQ ID No.1, and the base sequence of the coding gene is shown as SEQ ID No. 2. The application is the application of the coding gene in cultivating insect-resistant transgenic corn plants, the application in preparing recombinant insect-resistant protein Cry1ABn39 and the application of insect-resistant protein Cry1ABn39 in preparing insect-resistant agents. The invention increases the expression quantity of the insect-resistant protein in transgenic materials by artificial modification of the insect-resistant protein and optimization of plant expression vectors, and does not influence the original insect-resistant function and other agronomic characters of transgenic corns, thereby being capable of more easily obtaining the insect-resistant effect.)

1. An insect-resistant protein Cry1ABn39, the amino acid sequence of which is shown in SEQ ID No. 1.

2. A gene encoding the protein of claim 1.

3. The encoding gene of claim 2, wherein the base sequence of the encoding gene is shown in SEQ ID No. 2.

4. A method of producing a pest-resistant protein Cry1ABn39, comprising:

obtaining cells of a transgenic host organism comprising the gene of claim 2 or 3;

culturing cells of said transgenic host organism under conditions that allow the production of the insect-resistant protein Cry1ABn 39;

recovering the insect-resistant protein Cry1ABn 39.

5. The use of the coding gene of claim 2 in breeding insect-resistant transgenic corn plants.

6. An expression vector comprising the coding gene of claim 2.

7. The transgenic maize callus carrying the vector of claim 5.

8. Use of the encoding gene of claim 2 for preparing recombinant insect-resistant protein Cry1ABn 39.

9. Use of the insect-resistant protein Cry1ABn39 of claim 1 in the preparation of an insect-resistant agent.

10. The use of claim 8, wherein the anti-insect agent is a corn borer larval anti-insect agent.

Technical Field

The invention relates to the technical field of genetic engineering and molecular biology, in particular to an insect-resistant protein Cry1ABn39, an encoding gene and application.

Background

Bt (Bacillus thuringiensis) insecticidal proteins derived from Bacillus thuringiensis have highly specific poisoning activities against insects of the Lepidoptera, Coleoptera, Homoptera, Diptera, and the like. The proteins which are successfully cloned at present and applied to the development of transgenic crops mainly comprise Cry-type and Cyt-type genes for coding Insecticidal Crystal Proteins (ICPs) and Vip-type genes for coding vegetative insecticidal proteins (Vip). The three types of gene expression proteins have different insect resistance mechanisms and insect resistance. It is generally believed that the mechanisms of action of Cry proteins include processes of lysis, enzymatic activation, binding to receptors, intercalation and formation of channels and ion channels. Over the past several decades, several tens of Bacillus thuringiensis strains and 130 or more of their encoded insecticidal crystal proteins have been identified. The application of the compounds not only greatly reduces the dependence of chemical pesticides in grain production, promotes the guarantee of food safety, but also obviously increases the economic income of agricultural production. Therefore, the excavation and development of novel, safe and reliable insect-resistant genes become research and development hotspots in the field of molecular agriculture.

Cry1Ab gene for preventing and controlling lepidoptera corn borer is the preferred gene of transgenic corn. In 1993, Michael et al transform Cry1Ab gene to obtain Bt transgenic plant with good resistance to corn borer. In 1993, Koziel et al obtained insect-resistant transgenic maize by transferring Cry1A (b) gene into maize inbred lines. In 1996, Joachim W et al truncated and modified Cry1Ab to transform rice to obtain Bt-transformed rice, and the death rate of target pests reaches 100%. In 1998, Cheng et al transformed Cry1Ab and Cry1Ac genes into rice by Agrobacterium transformation according to the codon preferred by plants to obtain transgenic plants, and the R2 generation insect test shows 100% lethality within 5 days. With the application of genetic engineering techniques, transgenic Bt insect-resistant maize optimized by codons, such as MON810 and MON89034, is also commercially produced. In terms of cross-resistance, studies have shown that: the Cry1F resistant european corn borer line, capable of completing larval development on transgenic corn TC1507 expressing Cry1F protein, was not cross-resistant to Cry1Ab and Cry9C, with only a low level of cross-resistance to Cry1Ac (Pereira E J G, 2008). It is believed that domain i participates in pore formation, domain ii determines specific binding of the toxin to the receptor, and domain iii primarily regulates toxin activity.

The research of China on Cry1Ab insecticidal protein genes mainly focuses on the creation aspect of transgenic crops, such as: the Cnaphalium japonicum (2002) introduces two plasmids containing Cry1Ab gene and Bar gene into a corn inbred line by a cotransformation method, the frequency of 2 gene co-integration is 70%, and the genes are in linkage inheritance and expression, and most of transformed plants have high insect resistance. Yuan Ying et al (2007) adopts a particle gun method to introduce an insecticidal toxin protein gene (Cry1A) into the northeast spring corn inbred line iron 7922, and proves that the exogenous gene is integrated into a corn genome and can be stably inherited through PPT screening, molecular identification and ELISA detection of progeny plants, and a batch of corn borer resistant inbred lines are screened. In recent years, researches show that the modification of Bt gene sequences and the optimization of related regulatory elements on the molecular level can improve the expression amount of genes, and the researches are paid more and more attention by researchers.

Disclosure of Invention

The invention aims to provide an artificially modified Cry1ABn39 gene, wherein 46 new amino acids are inserted after the first amino acid of the N segment of the encoded protein, so that the protein is effectively prevented from being degraded by corn endogenous protease in corn cells.

The technical scheme of the invention is realized as follows:

the invention provides an insect-resistant protein Cry1ABn39, the amino acid sequence of which is shown in SEQ ID No. 1.

"insect-resistant" as used herein means toxic to crop pests. More specifically, the target insects are pests, such as, but not limited to, lepidopteran insects, including corn borer larvae, rice stem borers, spodoptera frugiperda, and the like.

The invention further protects the gene for coding the protein, the base sequence of the gene is shown as SEQ ID No.2, and the coding gene is designed and synthesized through the following ideas:

1) firstly, training is carried out according to the expression quantity information of the collected natural protein in the corn cells, a vector machine algorithm model is established, and the model is utilized to analyze and simulate the relationship between the stability of the natural Cry1Ab protein in the corn leaf cells and the N-terminal amino acid composition of the natural Cry1Ab protein;

2) according to the analysis result, firstly, the new sequence information structure of adding 46 amino acids behind the first amino acid at the N end of the amino acid sequence of the published natural Cry1Ab protein on Genbank is the most stable and is an amino acid sequence shown as SEQ ID No. 1;

3) according to an amino acid sequence shown in SEQ ID No.1, a Gene Designer is utilized to design a coding Gene of the Gene, the preference of the coding codon of the amino acid for a maize codon is selected in the first place, an AT-rich structural domain, a suspected intron structure, a continuously existing repetitive structural domain and a common endonuclease recognition site which appears in the Gene and possibly influences the construction of the Gene for a plant expression vector in the future commercial application process are removed through a Gene Designer built-in algorithm tool, and the Gene is optimized, wherein the optimized Gene sequence is shown in SEQ ID No. 2;

such sites include, but are not limited to: BamHI, BsaI, BstEII, ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, NcoI, NdeI, NheI, NotI, NsiI, PacI, PciI, PmeI, PmlI, PsiI, PstI, PvuI, PvuII, SacI, SacII, SalI, SfiI, SmaI, SpeI, SphI, SspI, StuI, Swai, XbaI, XhoI, XmaI, and XmnI cleavage sites;

4) the optimized gene sequence is completely synthesized by a chemical synthesis method, inserted into a cloning vector and stored.

The invention further protects a method for producing the insect-resistant protein Cry1ABn39, which comprises the following steps:

obtaining cells of a transgenic host organism comprising the above-mentioned gene;

culturing cells of said transgenic host organism under conditions that allow the production of the insect-resistant protein Cry1ABn 39;

recovering the insect-resistant protein Cry1ABn 39.

The invention further protects the application of the coding gene in cultivating insect-resistant transgenic corn plants.

The invention further protects an expression vector containing the coding gene.

Furthermore, the expression vector can be a commercial plant expression vector pCAMBIA 3301.

The invention further protects the transgenic maize callus carrying the vector.

The invention further protects the application of the coding gene in preparing the recombinant insect-resistant protein Cry1ABn 39.

The invention further protects the application of the insect-resistant protein Cry1ABn39 in preparing an insect-resistant agent.

As a further improvement of the invention, the insect-resistant agent is a corn borer larva insect-resistant agent.

The invention has the following beneficial effects: according to the artificially modified insecticidal protein Cry1ABn39, 46 new amino acids are added to the N segment, so that the structural stability of the protein in corn cells is greatly enhanced, and the protein is prevented from being degraded by corn endogenous protease, and therefore, a higher expression level can be obtained in the corn cells. And through insect-resistant experiments, the improved protein and the natural protein have no significant difference on the insecticidal toxicity of the larvae of the ostrinia nubilalis;

the invention modifies the protein established by a vector machine algorithm model of the relation between the amino acid sequence of the corn endogenous protein and the expression quantity, analyzes the stability of the Cry1Ab protein in transgenic plants thereof and predicts the modification scheme of an N-terminal sequence, combines the protein insecticidal toxicity analysis experiment result with the system experiments on a plurality of different levels such as the analysis of the population protein content of the corn transgenic plants, realizes the reasonable design of the natural Cry1Ab protein, obtains the novel insecticidal protein Cry1ABn39 protein which does not reduce the insecticidal toxicity but can be more effectively accumulated in transgenic corn leaf cells, and ensures that the transgenic corn material obtains higher insecticidal protein accumulation and the resistance to pests is improved.

The invention increases the expression quantity of the insect-resistant protein in transgenic materials by artificial modification of the insect-resistant protein and optimization of plant expression vectors, and does not influence the original insect-resistant function and other agronomic characters of transgenic corns, thereby being capable of more easily obtaining the insect-resistant effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of a plant expression vector;

FIG. 2 is a graph of the effect of protein engineering on total protein content in transgenic plants;

FIG. 3 is a graph showing the effect of protein modification on the expression level of herbicide resistance protein in transgenic plants;

FIG. 4 is a graph showing the effect of protein modification on the expression level of insect-resistant proteins in transgenic plants.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: n-terminal modification of insect-resistant protein Cry1Ab

1) A vector machine algorithm model established by a corn leaf histiocyte natural protein expression quantity and amino acid sequence composition relation model is used for predicting the relation between the stability of natural Cry1Ab protein in corn leaf cells and N-terminal amino acid composition thereof by machine learning under the development environment of python, the relation between the natural amino acid expression quantity and the N-terminal amino acid collected from a Genbank database is subjected to necessary library establishment, predictive variable (explanatory variable) data and output (dependent variable) data are split into a training data set and a testing data set, and a linear regression model is established. Predicting and obtaining an N-terminal artificial design sequence capable of obviously improving the protein expression quantity by using the linear regression model;

2) an exhaustive method generates about 1000 artificial sequences to simulate the change trend of the first amino acid (methionine) at the N terminal of the natural Cry1Ab protein and then insert 15-60 random amino acids into the first amino acid, namely the methionine, so that the Cry1Ab protein is subjected to heterologous expression level in corn mesophyll cells, and the sequences are ranked according to the predicted score of the expression amount, and 3 amino acid sequences which are possibly obviously improved for the expression of the Cry1Ab protein are obtained in total, namely N10, N39 and N121.

Wherein, the N10 amino acid sequence is shown as SEQ NO. 3;

an N39 amino acid sequence shown as SEQ NO. 4;

the N121 amino acid sequence is shown as SEQ NO. 3.

Example 2: analysis of insect resistance of modified insect-resistant protein

1) The method comprises the steps of carrying out prokaryotic expression on three amino acid sequence Cry1Ab proteins fused with N10, N39 and N121 and wild Cry1Ab protein by using an escherichia coli prokaryotic expression system, purifying a protein expression product by Sephadex G-200 gel column chromatography, and determining the toxicity of the corn borer.

2) Weighing 5g of Asiatic corn borer artificial feed, placing the feed in a culture dish, cutting the feed into 1-2mm slices, and soaking the slices in 50ng/ul protein liquid for 2 hours. 10 larvae of the ostrinia furnacalis which are hatched for 24 hours are inoculated into each culture dish. The test was run with the same volume of distilled water as the control. Each process set 5 replicates. Culturing in a climatic incubator with a temperature of 28 deg.C, a photoperiod of 16h:8h (L: D), and a relative humidity of 80%. The number of surviving larvae was investigated and recorded. The results are shown in table 1, and the fusion of the Cry1Ab proteins with the two amino acid sequences of N10 and N121 can reduce the toxicity of the Cry1Ab protein to the larvae of the corn borer. The toxicity of only N39 on the Cry1Ab protein in the predicted amino acid sequence was not reduced.

Table 1: toxicity to corn borer larvae

Example 3: synthesis of encoding gene of novel insect-resistant protein Cry1Ab protein

Finally determining a novel artificially designed insect-resistant protein Cry1Ab, namely a novel artificial insecticidal protein Cry1ABn39 (the amino acid sequence is shown as SEQ ID No. 1) with an N39 sequence of 45 amino acids added behind the first amino acid (methionine) of a natural protein according to the vector machine simulation result and the analysis of the detection experiment result of the toxicity of the larvae of the corn borer with the modified protein;

according to the amino acid sequence shown in SEQ ID No.1, a Gene Designer is utilized to design a coding Gene of the Gene, the coding codon of the amino acid is optimally selected by adopting the maize codon preference, and then an AT-rich domain, a suspected intron structure, a continuously existing repetitive domain and common endonuclease recognition sites which appear in the AT-rich domain and can influence the construction of a plant expression vector in the future commercial application process of the Gene are removed by a Gene Designer built-in algorithm tool, such as: BamHI, BsaI, BstEII, ClaI, EcoRI, EcoRV, HindIII, HpaI, KpnI, NcoI, NdeI, NheI, NotI, NsiI, PacI, PciI, PmeI, PmlI, PsiI, PstI, PvuI, PvuII, SacI, SacII, SalI, SfiI, SmaI, SpeI, SphI, SspI, StuI, Swai, XbaI, XhoI, XmaI and XmnI cleavage sites, and the optimized gene sequence is shown in SEQ ID NO. 2;

the gene sequences are completely synthesized by a chemical synthesis method, inserted into a cloning vector and stored.

Example 4: expression difference analysis of two insecticidal proteins Cry1Ab and Cry1ABn39 in corn transgenic plants

1) To evaluate the expression differences in transgenic maize plants for both proteins, the same plant expression vector backbone, i.e., the commercial plant expression vector pCAMBIA3301, was used as a unified evaluation. Digesting the two by using restriction endonucleases NcoI and BstEII to release a reporter gene GUS gene in the original vector, adding the same endonuclease recognition sites to two ends of coding genes of Cry1Ab and Cry1ABn39 proteins in a mode of adding the restriction endonuclease recognition sites to the 5' ends of amplification primers, digesting and treating by using the restriction endonucleases respectively to ensure that the generated sticky ends are connected into a commercial plant expression vector pCAMBIA3301 with the GUS gene removed, successfully constructing plant expression vectors pCAMBIA3301-Cry1Ab and pCAMBIA3301-Cry1ABn39, wherein the vector structure schematic diagram is shown in figure 1.

2) Transforming the plant expression vector into agrobacterium EHA105 by a freeze-thaw method, infecting maize immature embryos serving as plant receptor materials, co-culturing the infected maize immature embryos in a dark environment for 3 days, transferring the maize immature embryos into an antibacterial culture medium for 7 days, generating resistant callus through 35mg/L glufosinate ammonium resistance screening, differentiating the callus, and detecting positive plants through PCR (polymerase chain reaction) to obtain maize transgenic plants of two insecticidal proteins Cry1Ab and Cry1ABn 39.

3) 20 wild-type corn plants were used as a control group, and the obtained Cry1Ab transgenic corn plant 37 and Cry1ABn39 transgenic corn plant 44 were used as two experimental groups, each having 0.05g leaf and 3 leavesRepeating the above steps, grinding into powder in liquid nitrogen, adding PBS buffer (137mmol/L NaCl, 2.7 mmol/L KCl, 10mmol/L Na) at a ratio of 1:3(W/V)₂HPO₄，2mmol/L KH₂PO₄) And performing ice-bath until the mixture is completely melted. Vortexed for 30 sec; centrifuging at 15000g for 20min at 4 deg.C, and collecting supernatant as total soluble protein. The protein is quantitatively detected by a Coomassie brilliant blue staining method, and the change of the total protein content in the transgenic plant leaves of different insecticidal proteins is analyzed, and the result is shown in figure 2, and the total protein content in the transgenic plant leaves is not obviously different from that in a wild type.

4) The analysis result of the bar gene expression quantity by using the commercial PPT protein mouse monoclonal antibody is shown in figure 3, and the modification of the insect-resistant protein has no significant influence on the herbicide-resistant gene (which is also a screening marker gene). Elisa detection is carried out by using a mouse monoclonal antibody of a commercial Cry1Ab protein C-terminal epitope, the content of insect-resistant protein is determined, and the result is shown in figure 4, and the expression level of the insect-resistant protein of 44 transgenic corn plants of the modified insect-resistant protein Cry1ABn39 is obviously higher than that of 37 transgenic plants of the unmodified insect-resistant protein Cry1 Ab.

Compared with the prior art, the invention provides an artificially modified insecticidal protein Cry1ABn39, wherein 45 new amino acid sequences are inserted behind the first amino acid of the N segment of the protein, so that the modified protein is not easily recognized and degraded by endogenous protease in corn cells, and the expression product can be accumulated in the corn cells, thereby more easily obtaining a high-expression transgenic corn material. The in vitro insect-resistant experiment on the protein and the unmodified Cry1Ab protein shows that the two proteins have no significant difference in insecticidal toxicity to larvae of the corn borers. Therefore, transgenic corn with Cry1ABn39 protein that accumulates more readily in corn leaf cells is clearly more likely to gain an advantage in pest resistance.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<120> insect-resistant protein Cry1ABn39, encoding gene and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 2

<211> 2603

<212> DNA

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 2

ccatggcccc ggcggtgatg gcctcctccg ccaccacggt ggccccgttc caggggctga 60

agtccaccgc cggcctgccg atctcctgcc gctccggctc caccggcctg tcctccgtgt 120

ccaacggcgg caggatcagg tgcgacaaca acccaaacat caacgagtgc atcccgtaca 180

actgcctctc taacccggag gtcgaggtgc tcggcggtga gcgcatcgaa accgggtaca 240

cccccatcga catctccctc tccctcacgc agttcctgct cagcgagttc gtgccaggcg 300

ctggcttcgt cctgggcctc gtggacatca tctggggcat ctttggcccc tcccagtggg 360

acgccttcct ggtgcaaatc gagcagctca tcaaccagag gatcgaggag ttcgccagga 420

accaggccat cagcaggctg gagggcctca gcaacctcta ccaaatctac gctgagagct 480

tccgcgagtg ggaggccgac ccgactaacc cagctctccg cgaggagatg cgcatccagt 540

tcaacgacat gaacagcgcc ctgaccaccg ccatcccact cttcgccgtc cagaactacc 600

aagtcccgct cctgtccgtg tacgtccagg ccgccaacct gcacctcagc gtgctgaggg 660

acgtcagcgt gtttggccag aggtggggct tcgacgccgc caccatcaac agccgctaca 720

acgacctcac caggctgatc ggcaactaca ccgaccacgc tgtccgctgg tacaacacgg 780

gcctggagcg cgtctggggc ccggattcta gggactggat tcgctacaac cagttcaggc 840

gcgagctgac cctcaccgtc ctggacattg tgtccctctt cccgaactac gactcccgca 900

cctacccgat ccgcaccgtg tcccaactga cccgcgaaat ctacaccaac cccgtcctgg 960

agaacttcga cggtagcttc aggggcagcg cccagggcat cgagggctcc atcaggagcc 1020

cacacctgat ggacatcctc aacagcatca ccatctacac cgatgcccac aggggcgagt 1080

actactggag cggccaccag atcatggcgt ccccggtcgg cttcagcggc ccggagttta 1140

cctttcctct ctacggcacg atgggcaacg ccgctccaca acaacgcatc gtcgcccagc 1200

tcgggcaggg cgtctaccgc accctgtcct ccaccctgta ccgcaggccg ttcaacatcg 1260

gtatcaacaa ccagcagctc tccgtcctgg atggcactga gttcgcctac ggcacctcct 1320

ccaacctgcc ctccgctgtc taccgcaaga gcggcacggt ggattccctg gacgagatcc 1380

caccacagaa caacaatgtg ccaccgaggc agggtttttc ccacaggctc agccacgtca 1440

gcatgttccg ctccggcttc agcaactcct ccgtgagcat catcagggcc ccgatgttct 1500

cctggattca tcgcagcgcg gagttcaaca atatcattcc gtcctcccaa atcacccaaa 1560

tccccctcac caagtccacc aacctgggca gcggcacctc cgtggtgaag ggcccaggct 1620

tcacgggcgg cgacatcctg cgccgcacct ccccaggcca gatcagcacc ctccgcgtca 1680

acatcaccgc tcccctgtcc cagaggtatc gcgtcaggat tcgctacgcg agcaccacca 1740

acctgcaatt ccacacctcc atcgacggca ggccgatcaa tcagggtaac ttctccgcca 1800

ccatgtccag cggcagcaac ctccaatccg gcagcttccg caccgtgggt ttcaccaccc 1860

ccttcaactt ctccaacggc tccagcgttt tcaccctgag cgcccacgtc ttcaattccg 1920

gcaatgaggt gtacattgac cgcattgagt tcgtcccagc cgaggtgacg ttcgaagccg 1980

agtacgacct ggagagggcg caaaaggctg tcaatgagct gttcacgtcc agcaatcaga 2040

tcgggctgaa gaccgacgtg actgactacc acatcgacca agtctccaac ctcgtggagt 2100

gcctctccga tgagttctgc ctcgacgaga agaaggagct gtccgagaag gtgaagcacg 2160

ccaagaggct cagcgacgag aggaatctcc tccaggaccc caatttcagg ggcatcaaca 2220

ggcagctcga caggggctgg cgcggcagca ccgacatcac gatccagggc ggcgacgatg 2280

tgttcaagga gaactacgtg actctcctgg gcactttcga cgagtgctac ccaacctact 2340

tgtaccagaa gatcgacgag tccaagctca aggcttacac tcgctaccag ctcaggggct 2400

acatcgaaga cagccaagac ctggagattt acctgatccg ctacaacgcc aagcacgaaa 2460

ccgtcaacgt gcccggtact ggttccctct ggccgctgag cgcccccagc ccgattggca 2520

agtgtgccca ccacagccac cacttctccc tggacatcga cgtgggctgc accgacctga 2580

acgaggactt tcggtaggtg acc 2603

<210> 3

<211> 864

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 3

Met Ala Pro Ala Val Met Ala Ser Ser Ala Thr Thr Val Ala Pro Phe

1 5 10 15

Gln Gly Leu Lys Ser Thr Ala Gly Leu Pro Ile Ser Cys Arg Ser Gly

20 25 30

Ser Thr Gly Leu Ser Ser Val Ser Asn Gly Gly Arg Ile Arg Cys Asp

35 40 45

Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn

50 55 60

Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr

65 70 75 80

Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe

85 90 95

Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp Gly

100 105 110

Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu Gln

115 120 125

Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser

130 135 140

Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe

145 150 155 160

Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu Met

165 170 175

Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile Pro

180 185 190

Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr Val

195 200 205

Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe

210 215 220

Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn

225 230 235 240

Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg Trp

245 250 255

Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp Trp

260 265 270

Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu Asp

275 280 285

Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg

290 295 300

Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu

305 310 315 320

Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser

325 330 335

Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile Tyr

340 345 350

Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln Ile Met

355 360 365

Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr

370 375 380

Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu

385 390 395 400

Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro

405 410 415

Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly Thr

420 425 430

Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr Arg

435 440 445

Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn

450 455 460

Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser

465 470 475 480

Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg Ala

485 490 495

Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile Ile

500 505 510

Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr Asn Leu

515 520 525

Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp

530 535 540

Ile Leu Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val Asn

545 550 555 560

Ile Thr Ala Pro Leu Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala

565 570 575

Ser Thr Thr Asn Leu Gln Phe His Thr Ser Ile Asp Gly Arg Pro Ile

580 585 590

Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn Leu Gln

595 600 605

Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe Ser

610 615 620

Asn Gly Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser Gly

625 630 635 640

Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val Thr

645 650 655

Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu

660 665 670

Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp

675 680 685

Tyr His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu

690 695 700

Phe Cys Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala

705 710 715 720

Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg

725 730 735

Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile

740 745 750

Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu

755 760 765

Leu Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile

770 775 780

Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr

785 790 795 800

Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala

805 810 815

Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu

820 825 830

Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His His Phe

835 840 845

Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Phe Arg

850 855 860

<210> 3

<211> 40

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 3

Ser Thr Ala Ser Pro Ala Pro Thr Leu Lys Thr Ile Arg Cys Ser Thr

1 5 10 15

Ala Leu Val Ser Thr Thr Ala Arg Val Ala Pro Phe Ala Gly Arg Lys

20 25 30

Asn Met Ala Ser Leu Val Gly Arg

35 40

<210> 4

<211> 40

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 4

Ala Pro Thr Val Met Ala Ser Ser Ala Thr Ala Val Ala Pro Phe Gln

1 5 10 15

Gly Leu Lys Ser Thr Ala Thr Leu Pro Val Ala Arg Arg Ser Thr Thr

20 25 30

Ser Leu Ala Lys Val Ser Asn Gly

35 40

<210> 5

<211> 58

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 5

Ala Thr Leu Ala Thr Ala Val Pro Val Ala Phe Gln Gly Arg Thr Lys

1 5 10 15

Thr Ile Arg Cys Thr Val Ser Thr Ala Pro Lys Thr Leu Ala Lys Val

20 25 30

Ser Asn Ser Thr Ser Pro Thr Ala Val Ala Pro Ala Gly Arg Lys Asn

35 40 45

Ala Ser Val Gly Arg Ser Thr Ser Ala Pro

50 55