CN1259421C - Bt-cry1 Ah gene with high toxicity for lepidoptera and its expression product - Google Patents

Abstract

The invention relates to "Bt crylAh gene with high toxicity to Lepidoptera insects", which belongs to the technical field of biological control. The invention relates to a Bt crylAh gene and a partial sequence thereof which are resistant to Lepidoptera pests, and protein and polypeptide sequences encoded by the gene and the partial sequence. Further, the present invention also relates to the application of these genes and their partial sequences and the proteins and polypeptides encoded by them in the biological control of Lepidoptera pests.

Description

Btcry1Ah gene and its expression product highly virulent to Lepidoptera

Technical field of the present invention:

The invention belongs to the technical field of biological control. Further, the present invention relates to the Btcry1Ah gene and a partial nucleotide sequence thereof, which are highly toxic to Lepidoptera pests, and proteins and polypeptides encoded by the gene and the partial sequence thereof.

Research background of the present invention:

Bacillus thuringiensis (Bt for short) is a Gram-positive spore-forming soil bacterium that is widely distributed in areas such as soil, insect corpses, plant root circles, and granary dust. As we all know, it can produce protein crystals in the spore formation stage, and has poisonous activity against insects such as Lepidoptera, Coleoptera, Diptera, Hymenoptera, Trichophaera, Homoptera, and even animals such as nematodes. It is safe and harmless to non-target organisms such as humans, higher animals, and plants, and has been widely used in the prevention and control of agricultural, forestry, and sanitary pests.

In 1981, Schnepf and Whiteley cloned the first Bt insecticidal protein gene cry1Aa, and completed the nucleotide sequence analysis in 1985. The gene is active against Lepidoptera pests. Subsequently, active bacterial strains and genes to pests such as Coleoptera and Diptera have been discovered and isolated and cloned (Krieg, A.et al, J.Appli.Entomology, 1983, 96:500-508; Ward, ES, and DJ Ellar. 1986. J. Mol. Biol. 191: 1-11. Herrnstadt, C. et al, Gene, 1987, 57(1): 37-46; Payne, J., KENarva, and J. Fu. December 1996 .US Patent 5,589,382.). With the rapid development of biotechnology, the commercialization of Bt genetically engineered bacteria and insect-resistant plants has achieved great success. All countries have increased investment in order to obtain new high-virulence and new active Bt strains and genes. As of the end of October 2004, a total of 323 Bt insecticidal protein genes have been discovered and published worldwide (see http://www.biols.susx.ac.uk/home/Neil Crickmore/Bt/list.html ) .

In 1996, the world's first transgenic insect-resistant plant was approved for use in the United States, and the main part of the gene it used came from Bt cry1Ac. In the next few years, GMOs such as cry1Ab-transferred insect-resistant corn and cry3Aa-transferred insect-resistant potatoes came out one after another. By the end of 2003, the planting area of insect-resistant cotton in the United States accounted for 73% of the total cotton planted, and corn accounted for to 40% (Clive James, International Service for the Acquisition of Agri-Biotech Application, preview, No.30, 2003). In China, since the official promotion of insect-resistant cotton containing the cry1Ac/1Ab gene in 1998, the cumulative planting area has reached 90 million mu, and the planting area in 2004 alone was as high as 37 million mu. Also approved for commercialization in my country is transgenic insect-resistant poplar, which uses almost the same gene as insect-resistant cotton. At the same time, the insect-resistant transgenic plants that are being released in the environment by domestic research institutes include rice (cry1Ac/1Ab or cry1Ab) and corn (cry1Ac/Ab). However, the genes used in insect-resistant engineering plants, whether commercialized or in the research and evaluation stage, have the disadvantage of a single type. Therefore, if these insect-resistant crops are widely promoted, there will be risks of reducing pest refuges and increasing drug resistance too quickly. An effective way to solve this problem is to find new high virulence genes or gene combinations as new replacements and supplements. The second-generation insect-resistant cotton of Monsanto Company of the United States has adopted the cry2Ab gene that is highly toxic to cotton bollworm (Bruce E. Tabashnik et al., APPLIEDAND ENVIRONMENTAL MICROBIOLOGY, Aug.2002, p.3790-3794; Nicholas, D. et al, Transgenic Plant and Insect Pests Biocontrol, John Wiley & Sons Press, USA, 1997, 1-18).

Therefore, screening and cloning new, highly toxic Bt insecticidal protein genes will enrich insecticidal gene resources, provide new gene sources for the development of genetically engineered bacteria and insect-resistant plants, and improve the insect-resistant effect of Bt transgenic products , and is expected to reduce the risk of pest resistance to Bt toxins and avoid new ecological disasters, which has important economic, social and ecological benefits.

Contents of the present invention:

Purpose of the present invention:

Aiming at the shortcomings of the Bt preparations and transgenic products used in the world to prevent and control Lepidoptera pests, the single gene variety, easy resistance of pests, narrow insecticidal spectrum and low toxicity, the present invention isolates and clones Bt strains from domestic Bt strains. The new gene cry1Ah, the expression product of this gene is significantly more toxic to various Lepidoptera pests than the currently known Cry protein. This new gene can be applied to the transformation of microorganisms and plants, making them highly toxic to related pests, and overcoming and delaying the emergence of pest resistance to engineering bacteria and transgenic plants.

Technical scheme of the present invention:

1. Identification of the cry gene in the starting strain Bt BT8 strain

Adopt Kuo (Kuo and Chak, Appl.Environ.Micro.1996, 62:80-86) and Song Fuping's PCR-RFLP identification system to domestic Bt isolates (Song Fuping, Zhang Jie, Huang Dafang, etc., "Chinese Agricultural Science", 1998, Volume 31, Issue 3, Pages 13-18) conducted cry gene analysis and found that known genes such as cry1Aa, cry1Ba cry2Aa, and cry2Ab existed in the strain, and a new gene cry1X that could not be named was also discovered.

2. Cloning of cry1Ah gene in BT8 strain

The new cry gene in the strain was isolated and cloned by rapid cloning method.

According to Narva (Narva, K.E.et al, EP0462721 A2,1991,8) etc., extract plasmid DNA from the Bt BT8 bacterial strain (this bacterial strain comes from the biotechnology laboratory of Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and can provide to the public), The plasmid DNA was partially digested with Sau3AI enzyme, and the 4-7kb DNA fragment was recovered from the gel. After purification, it was ligated with the pUCP18 vector digested by BamHI and treated with alkaline phosphatase (Schweizer HP, Gene, 1991 Jan2; 97 (1): 109-21.), after transforming Escherichia coli JM107 (purchased from U.S. NEBiolab company, 32 Tozer Road Beverly, MA01915-5599, USA), use the cry1 gene universal primer K5un2/K3un2 to the positive transformant that can grow normally on the antibiotic Amp Carry out PCR amplification identification, and screen the positive transformants containing Bt cry gene. The primers are:

Forward primer: TCCTGCAGTTGACTTCAAATAGG;

Reverse primer: CAGTCGACTCATCCGATAAACACGCCAC’

The pHT59 was subcloned by the procedures and conditions shown in Fig. 6 . DNA sequence analysis was carried out on the recombinant plasmid pHT59 obtained by screening, and the results showed that the plasmid contained the full-length DNA fragment of the cry1Ah gene. The full sequence of the gene is shown in SEQ ID NO 1, containing a total of 3549bp; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO 2, the protein is composed of 1182 amino acids, the molecular weight is 134kDa, and the isoelectric point is pl 4.985. The Btδ-endotoxin gene International Nomenclature Committee named the gene cry1Ah1.

3. Construction of Shuttle Expression Vector

Design a pair of primers according to the 3549bp nucleotide sequence of the cry1Ah1 full-length gene, and add a restriction enzyme site at the 5' end of the primers for the connection of the gene and the expression vector. The primer sequences are as follows:

Forward primer: 5'CG GGATCC ATGAAAAACAGTATCAA3'

BamHI

Reverse primer: 5'ACGC GTCGAC CTATTCTCTCATAAGGAG 3'

SalI

The 3549bp PCR product was digested with BamHI and SalI and cloned into the Bt-E.coli shuttle plasmid pSXY422 (7.3kb) containing the Bt replicon. The plasmid was transformed from pUC18, added the replicon of the Bt plasmid, the vegetative expression promoter Pcry3Aa of the Bt cry gene, and the multiple cloning site from the pET21b plasmid, and became a shuttle expression vector (the plasmid was obtained from the Plant Protection Research Institute of the Chinese Academy of Agricultural Sciences biotechnology laboratories, available to the public). The obtained recombinant expression vector plasmid was named pSXY422-1Ah. Using the transformation method of Lereclus et al. (Lereclus, D. et al, FEMS Microbiology Letters, 1989, 60: 211-217), pSXY422-1Ah was used to transform the Bt crystal-free mutant strain ^HD73- (the strain came from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences Biotechnology laboratory, available to the public). A variety of molecular tests were performed on the positive transformants that could grow on the erythromycin-resistant plate to prove that the transformation was successful. The transformant was named engineering bacteria Biot1Ah. The bacterium can be stably inherited in the Bt crystal-free mutant strain ^HD73- , and the stability is greater than 90%.

4. Cultivation and observation of engineered bacteria

Adopt GT culture medium (recipe: liquid GT: add 10ml 0.8% CaCl ₂ per liter, 10ml G-Tris salt (0.025% FeSO ₄ .7H ₂ O, 0.05% CuSO ₄ .5H ₂ O, 0.05% ZnSO ₄ . 7H ₂ O, 0.5% MnSO ₄ .H ₂ O, 2.0% MgSO ₄ ), 10ml 20% (NH ₄ ) ₂ SO ₄ , 10ml 5% K ₂ HPO ₄ , 10ml 20% glucose, 50ml 1M Tris-HCl, pH7. 5. 1.5g yeast extract, pH7.4; solid GT: add 1.3% agar to liquid GT), 1/2LB medium (formula: liquid LB: Tryptone 1%, yeast extract 0.5%, NaCl 1% , pH7.0; solid LB: add 1.3% agar to the liquid medium) to cultivate Biot1Ah engineering bacteria respectively, and observe the results with scanning electron microscope and optical microscope. The results showed the presence of bipyramidal crystals in Biot1Ah, proving the normal expression of this gene.

5. Determination and expression study of the 5'-terminal active region of cry1Ah1 gene

According to the fact that the active region of other cry1A genes is larger than 1.8kb, primer B. 2001bp partial gene primers were designed:

Forward primer: 5'CG GGATCC ATGAAAAACAGTATCAA 3'

BamHI

Reverse primer: 5'ACGC GTCGAC TGATCAATATGATAATCCG 3'

SalI

The 2001bp PCR product was digested by BamHI and SalI and cloned into the E. coli expression vector pET21 (purchased from Novagen Inc, 601 Science Drive Madison, Wi 53711, USA), and the obtained recombinant expression vector plasmid was named pET1Ah-2. Escherichia coli BL21(DE3) was transformed with pET1Ah-2 (Dubendorff, J.W. and Studier, E.W.1991, J.Mol.Biol.219, 45-59.), and a 75kD polypeptide was induced by IPTG.

6. Determination of insecticidal biological activity

The Lepidoptera pests used include: diamondback moth (Plutella xylostella) as the second instar larvae; cotton bollworm (Helicoverpa armigera), Asian corn borer (Ostrina furnacalis) and rice stem borer (Chilo supperssalis) as the first instar larvae. The above-mentioned test insects are standard laboratory breeding populations.

There is also the soybean borer (Leguminivora glycinivorella): trapping adults in naturally planted soybean fields, and laying eggs and hatching 1 to 2 larvae indoors.

The individual test and combined test are carried out with different concentrations of Bt poisonous protein as samples, and the time is 4-7 days. The experimental data were processed according to the standard.

Beneficial effects of the present invention:

The isolated and cloned Bt cry1Ah gene of the present invention and a partial sequence thereof and their gene expression products can produce strong virulence to Lepidoptera pests, which is stronger than the genes of similar natural strains found in the world at present. Through the combination of cry1Ah gene and cry1Ab, cry1Ba, cry2Ab and other gene expression products, the insecticidal spectrum against Lepidoptera, Coleoptera and Diptera pests can be expanded. By applying it to transforming microorganisms and plants, making them exhibit toxicity to related pests, it can overcome or delay insect resistance to engineering bacteria and transgenic plants.

Description of drawings:

Figure 1 shows the PCR amplification product and RFLP of BT8 cry1 gene.

Where M stands for pUCDNA Mixed Marker (1116, 883, 692, 501, 489, 404, 331, 242, 190, 147, 111, 110bps), 1 is Kun3 primer PCR amplification product 1.4kb, 2 is 1/PstI and EcoRI Restriction digestion, 3 is 4/PstI and XbaI digestion, 4 is the amplification product of Kun2 primer 1.6kb.

Fig. 2 is the PCR-RFLP identification of the recombinant plasmid pHT59 containing the target gene.

Among them, M represents pUC DNA mixed marker, 1 is the amplification product of Kun2 primer/PstI+XbaI, and 2 is the amplification product of Kun3 primer/PstI+EcoRI.

Figure 3 is the single enzyme digestion analysis of pHT59.

Among them, 1, 2, 3, 4, 5, 6 and M respectively represent: pHT59/EcoRI, HindIII, KpnI, PstI, SacI, XbaI and λDNA/Eco130I marker (19.1, 7.7, 6.2, 4.2, 3.4, 2.7, 1.9, 1.5, 0.9, 0.4kb).

Figure 4 is the double enzyme digestion analysis of the pHT59 plasmid.

Among them, 1, 2, 3, 4, 5, 6, 7 and M represent pHT59/EcoRI and XbaI, pHT59/EcoRI and KpnI, pHT59/EcoRI and HindIII, pHT59/EcoRI and PstI, pHT59/EcoRI and SacI, pHT59/ SacIand PstI, pHT59 plasmid and λDNA/EcoO130I marker.

Fig. 5 Enzyme digestion analysis of subcloned plasmids pHT59-32, pHT59-15, pHT59-22.

Among them, 1, 2 represent pHT59-15/HindIII, 3, 4 represent pHT59-32/HindIII, 5, 6 represent pHT59-22/HindIII, M is λDNA/EcoO130I marker.

Fig. 6 is a flowchart of the subcloning of cry1Ah gene/pHT59.

Fig. 7 shows restriction analysis of cry1Ah full-length gene shuttle expression vector pSXY422-1Ah.

Among them, 1 and 2 are PCR amplification products of cry1Ah full-length gene, 3 is the shuttle vector pSXY422/BamHI andSalI, 4 is pSXY422-1Ah/BamHI and SalI, and M represents λDNA/EcoO130I marker.

Fig. 8 is PCR amplification detection of 2.0kb fragment of cry1Ah gene.

Among them, 1 and 2 are E.coli expression vector pET21b/BamHI and SalI, 3 and 4 represent the 2.0kb PCR amplification product of the 5' end of the cry1Ah gene, and M represents the λDNA/EcoO130I marker.

Fig. 9 is the enzyme digestion analysis of the recombinant plasmid pET1Ah-2 containing the 2.0 kb fragment of the cry1Ah gene.

Among them, 1 and M stand for pET1Ah-2/BamHI and SalI and λDNA/EcoO130I marker respectively.

Figure 10 is the SDS-PAGE detection of the 2.0kb fragment of cry1Ah gene expressed in E.coli.

Among them, 1 and 2 represent inclusion body protein components formed by expressing cells, 3 is soluble protein components, 4 is negative control bacteria (BL21: pET21b), and M represents molecular weight markers (212, 116, 97, 66, 40kD).

Figure 11 shows the expression of various cry genes in BT8 at different culture times.

Among them, 12, 18, 24, 30, and 36 respectively represent the bacterial culture time (hours), and M represents the molecular weight marker (212, 116, 97, 66, 40kD).

Figure 12 is the expression of cry1Ah gene in engineering bacteria Biot1Ah at different culture time.

Among them, 18, 24, 30, 36, and 48 represent the bacterial culture time (hours), and M represents the molecular weight marker (212, 116, 97, 66, 40kD).

Fig. 13 is the scanning electron microscope result of Cry1Ah protein crystal (engineering bacteria Biot1Ah).

Fig. 14 is a homologous phylogenetic tree of the nucleotide sequences of the full-length cry1Ah gene and other cry1 genes.

Figure 15 is a homologous phylogenetic tree of the nucleotide sequence of cry1Ah 2.0kb gene and other cry1 genes of the same length.

Figure 16 is a homologous phylogenetic tree of the full-length amino acid sequences of the Cry1Ah protein and other Cry1-like proteins.

Figure 17 is a homologous evolution comparison of the N-terminal 667 amino acid sequences of the Cry1Ah protein and other Cry1-like proteins.

Specific embodiments of the present invention:

Examples of the present invention are described below. It should be noted that the embodiments of the present invention are only illustrative but not limiting to the present invention.

The genotype identification of embodiment 1, BT8 bacterial strain

Extract plasmid DNA from Bt bacterial strain BT8 according to the method of Narva (Narva, K.E.ct al, EP0462721 A2,1991). Using the plasmid DNA of the B-8-G strain as a template, the 3' end primers (K5un2 and K3un2) and the 5' end primers (K5un3 and K3un3) of cry1 were used for PCR amplification, and the PCR cycle parameters were 94°C for 1 minute, 54 °C for 1 minute, 72°C for 3 minutes, 32 cycles, 72°C for 10 minutes. Two PCR products of 1.6kb at the 3' end and 1.4kb at the 5' end of the cry1 gene were obtained respectively. The DNA fragments were extracted and purified with chloroform and then digested with restriction enzymes. Gel electrophoresis was used to analyze the polymorphism of the digested fragments (Fig. 1). The analysis results (see Table 1) showed that the BT8 strain contained cry1Aa, cry1Ba, cry2Aa, cry2Ab and an unknown new cry1 gene (cry 2 gene results are omitted).

Table 1 PCR amplification products and restriction enzyme length polymorphisms of cry1 gene in BT8 strain Gene PCR amplification product (K5un3/K3un3) (bp) EcoRI and PstI fragments (bp) cry1Aa 1463 726, 493, 244 cry1Ba NP cry1Ax 1460 940, 520 Gene PCR amplification product (K5un2/K3un2) (bp) XbaI and PstI fragments (bp) cry1Aa 1635 1117, 518 cry1Ba 1686 1015, 655, 16 cry1? 1630 780 540 310

The cloning of embodiment 2, BT8 bacterial strain new gene

It was identified that the BT8 strain contained an unknown new cry1 gene, which was isolated and cloned by the following method. The BT8 plasmid was partially digested with Sau3AI, and a 4-7kb fragment was recovered, ligated with the vector pUCP19 digested with BamHI to transform JM107, and a positive transformant ETG59 containing the new cry1 gene of the BT8 strain was obtained by PCR amplification and RFLP analysis (figure 2). , named the recombinant plasmid pHT59.

According to the restriction analysis of the existing cry1 gene, select EcoRI, HindIII, KpnI, PstI, SacI, XbaI, etc. to carry out a single enzyme digestion analysis on pTG59 (Fig. 3). The results show that the foreign fragment cloned on pHT59 is 6.9kb. After EcoRI and XbaI, EcoRI and KpnI, EcoRI and HindIII, EcoRI and PstI, EcoRI and SacI, SacI and PstI double enzyme digestion analysis (accompanying drawing 4), the physical map of the large fragment of the gene was constructed. According to the results of various enzyme digestion analysis, HindIII fragments were selected for subcloning. pHT59 was completely digested by HindIII into three fragments of 6.7kb (2.2kb and pUCP19 4.5kb), 3.2kb and 1.5kb, respectively. HindIII digested pBlueScriptSK ⁺ ligation, 67kb fragments were self-ligated, transformed into Escherichia coli, and three subclones were obtained through plasmid analysis and screening, and the recombinant plasmids contained were named pHT59-32, pHT59-15, and pHT59-22 (with accompanying drawings) 5). Accompanying drawing 6 is this subcloning flowchart.

The sequences of the three subclones were determined, and the homologous sequences were compared with other cry1 genes, which indicated that the gene was a new full-length gene. The coding region of the gene is 3549bps (SEQ ID NO 1), encoding a protein (SEQ ID NO 2) of 1182 amino acids, with a molecular weight of 134kDa and an isoelectric point of pH4.985. The gene was named cry1Ah1 by the International Nomenclature Committee of Btδ Endotoxin Genes.

Example 3, construction of cry1Ah full-length gene shuttle vector

In order to facilitate cloning and expression, a pair of specific primers containing BamHI and SalI restriction sites were designed according to the full-length nucleotide sequence of the cry1Ah1 gene coding region. The PCR amplification conditions were 94°C for 1 minute, 54°C for 1 minute, 72°C for 3 minutes, 32 cycles, and 72°C for 10 minutes, using high-fidelity pfu polymerase for amplification. A 3.5kbcry1Ah full-length fragment was obtained, as shown in the first and second samples of A in Fig. 7 . After the complete reaction of BamHI and SalI double enzyme digestion, perform electrophoresis, recover the fragment from the gel, and perform ligation reaction with the Bt-E.coli shuttle expression vector pSXY422 treated by the same enzyme digestion, 12 ℃ for 12 hours; 2 μL of the product was transformed into E.coli recipient strain JM110, and positive recombinant plasmids were screened with Amp resistance plates. The obtained positive recombinant plasmids were respectively inoculated into liquid LB test tubes, cultured at 37°C and 230 rpm for 12 hours, the plasmids were extracted by the commonly used alkaline hydrolysis method, and the single and double enzyme digestion reactions of BamHI and SalI were carried out, and identified by gel electrophoresis. The recombinant expression clone pSXY422-1Ah of the 3.5kb full-length fragment of the cry1Ah gene, B in Figure 7 is the result of double enzyme digestion, of which No. 3 is the pSXY422 empty vector and No. 4 is pSXY422-1Ah.

Embodiment 4, the construction of Bt engineering bacteria Biot1Ah

The recombinant expression clone pSXY422-1Ah from JM110 was transformed into the E.coli methylation mutant strain SCS110 by electric shock method. The electric shock conditions were: 2500V, 400Ω, 25μF, 100μL competent cells, and 1μL plasmid DNA. Incubate at 37°C for 1 hour, 100 rpm; extract the demethylated pSXY422-1Ah plasmid from SCS110.

The Bt crystal-free mutant strain HD-73 ^- competent was prepared, and the demethylated pSXY422-1Ah plasmid was introduced into it by electric shock transformation. Electric shock conditions: 2200V, 1000Ω, 25μF, 100μL competent cells, 1μL plasmid DNA. Incubate at 30° C. for 2 hours, 100 rpm; select positive transformants with 25 μg/mL Ery (erythromycin) plate. After extracting the plasmid, restriction analysis, PCR amplification and other molecular tests, a Bt-positive transformant containing the pSXY422-1Ah expression plasmid was obtained, and the transformant was named the engineering bacterium Biot1A.

Embodiment 5, the cultivation, observation and detection of Bt engineering bacteria Biot1Ah

Activate engineering bacteria Biot1Ah (30°C, 12 hours); inoculate 1% in 1LGT medium, add erythromycin, the final concentration is 25 μg/mL, 30°C, 230rpm, shaking culture for 20-48 hours; Take 1mL of the culture medium every 6 hours for spatiotemporal research on protein expression; meanwhile, culture the starting strain BT8 as a reference. A small amount of culture was taken for light microscopy to observe cell lysis, release of spores and crystal formation. When 40% of the vegetative cells are lysed, stop the cultivation, collect the bacteria by centrifugation, add 100mL 1M NaCl/10mM Tris·Cl (pH7.5) solution to wash twice, and wash three times with sterile water to remove fermentation secondary metabolism Centrifuge at 6,000 rpm for 10 minutes at 4°C (these cultures can be stored at -20°C for later use). The above cell crystal mixture was taken for observation and recording by scanning electron microscope. The results (Fig. 13) show that the cry1Ah gene can be expressed normally in the Bt crystal-free mutant strain HD- ^73- , forming bipyramidal crystals unique to the cry1 gene.

Take 20 mg of the above-mentioned cell crystal mixture, suspend in 100 μL water, add 25 μL 0.5N NaOH, and react at 25°C for 5 minutes; add 65 μL 3×sample buffer, boil at 100°C for 5 minutes, centrifuge to remove the precipitate; perform SDS-PAGE electrophoresis detection respectively, The results are shown in Figures 11 and 12. Figure 11 shows that: in the starting strain BT8, the protein of about 130kD can be detected from 18 hours to 36 hours, and can exist and accumulate stably, and the number of crystals is relatively large. In the engineered bacteria Biot1Ah, the 134kDa Cry1Ah protein (Fig. 12) can be detected after 24 hours, and it accumulates continuously as the culture time prolongs, forming a considerable number of stable bipyramidal crystals (Fig. 13). Same as natural strain. The results of continuous subculture and dilution plating test show that the genetic stability of the engineering bacteria is above 98%.

Example 6, cloning, expression and detection of 2.0kb fragment of cry1Ah gene active region

Cloning: According to the nucleotide sequence of cry1Ah1 gene from its initiation codon ATG to 2001bp in the 5'-section, a pair of specific primers containing BamHI and SalI restriction sites were designed. The PCR amplification conditions were 94°C for 1 minute, 54°C for 1 minute, 72°C for 2 minutes, 30 cycles, and 72°C for 10 minutes, and the reaction enzyme was pfu polymerase. Amplify and obtain the 2.0kbcry1Ah active region fragment (see the 3rd and 4th samples in the accompanying drawing 8), after the complete reaction of BamHI and SalI double enzyme digestion, carry out electrophoresis, recover this fragment from the gel, and through the same enzyme digestion treatment The E.coli expression vector pET21b was used for the ligation reaction, 18°C for 4 hours, and 2 μL of the ligation product was taken to transform the E.coli recipient strain BL21, and the positive recombinant plasmid was screened with the Amp resistance plate. The obtained positive recombinant plasmid was inoculated in an LB test tube for 12 hours at 37°C and 230 rpm, and the plasmid was extracted, and subjected to single and double enzyme digestion reactions of BamHI and SalI, respectively, to obtain a positive clone pET1Ah2 containing a 2.0kb fragment of the cry1Ah gene, as shown in Figure 9 It is the result of the double enzyme digestion test.

Induced expression: Activation positive clone BL21 (pET1Ah2) (37°C, 12 hours); 1% was inoculated in 1L LB medium, 37°C, 230rpm, shaking culture until OD ₆₀₀ was 0.5-0.8, (about 2 hours); The inducer IPTG, with a final concentration of 0.7mM, 150rpm, was induced at 20°C for 18 hours at low temperature; the bacteria were collected by centrifugation, and suspended by adding 50mL 10mM Tris Cl (pH8.0); Interval = 0.5 sec), treated for 10 minutes, and the reaction was controlled at 0°C; 4°C, 12,000 rpm, centrifuged for 10 minutes; collected supernatant and precipitate, respectively, for SDS-PAGE electrophoresis detection.

SDS-PAGE electrophoresis: treatment of the above protein samples: Take 100 μL of supernatant or suspension of inclusion body pellet in 100 μL of 10 mM Tris Cl buffer, add 50 μL of 3x sample buffer (25 μL of loading buffer + 75 μL of β-mercaptoethanol), Boil at 100°C for 5 minutes, centrifuge to remove the precipitate; add the samples to the stacking gel in turn, electrophoresis at 4°C; complete the staining, decolorization, gel scanning and data recording analysis. The expression product of 2.0kb in the active region of Cry1Ah is 75kD, which is consistent with the predicted results. The 2001kb partial nucleotide sequence of the cry1Ah gene is shown in SEQ ID NO 3, and the encoded polypeptide, which is half of Cry1Ah, has a sequence of 667 amino acids in total as shown in SEQ ID NO 4.

Embodiment 7, Cry1Ah is to the toxicity determination of several pests

1. Plutella xylostella: Weigh cabbage leaves and soak the leaves. Take 100g of fresh leaves and soak the Cry1Ah full-length protein in different sample dilution concentrations for 10 seconds. After taking it out, let it dry naturally at room temperature and place it in a wide-mouth bottle. Receive 20 2nd instar larvae, 25°C, daily light:dark ratio of 12:12 hours, set Cry1Ac and other different Bt Cry proteins as controls, and investigate the results for 96 hours.

2. The first-instar larvae were used for the bioassay of cotton bollworm, Asian corn borer, and rice stem borer. The Cry1Ah full-length protein sample was designed with a suitable concentration gradient according to the pre-test results, and the ratio of 10g artificial feed/mL protein sample was evenly mixed, and packed Incubate in finger tubes, 24-well plates and other vessels at 25-26°C, investigate at 96, 120, and 168 hours respectively, and calculate LC ₅₀ .

3. Soybean pod worm: put 3,000 adults caught in the field (the ratio of male to female is close to 1:1) and raise them in a soybean field (with pods) covered with a net in advance, and let them lay eggs naturally. 2 larvae were used for bioassay.

The samples were tested with different concentrations of Bt Cry1Ah full-length protein. Soak the green beans in the diluted sample to be tested for 10 seconds, dry them, put them into the peeled pods, then put the pods into the bioassay bottle, inoculate 20 worms per bottle, and repeat 3 times for each treatment. Incubate at 25°C for 24, 48, and 96 hours to investigate the death of soybean borer. Calculation of mortality, adjusted mortality.

See Table 2-Table 5 for the biological activity assay results of the full-length Cry1Ah protein.

Table 2 Insecticidal activity of Cry1Ah protein against second instar larvae of diamondback moth (Plutella.Xylostella) sample Concentrationug/ml Number of test insects Insect count % just% LC _50ug /ml CKCry1Ah 12.86.43.21.60.80.4 60606060606060 2595546291811 3.3398.3391.6776.6748.333018.33 98.2791.3875.8746.5527.5915.52 1.39(1.15-1.67)

Table 3 Insecticidal activity of Cry1Ah protein on first instar larvae of cotton bollworm (Helicoverpa armigera) sample Concentrationug/ml test insect Insect count % just% LC _50ug /ml CKCry1Ah 4512.86.42.561.60.8 48484848484848 0332823191411 0.0068.7558.3347.9239.5829.1722.92 68.7558.3347.9239.5829.1722.92 6.06 (3.99-11.94)

Table 4 Insecticidal activity of Cry1Ah protein against first instar larvae of Chilo supperssalis sample Concentration g/g test insect Insect count % just% LC ₅₀ g/g CKCry1Ah 20105210.5 50505050505050 2484238363115 4.0096.0084.0076.0072.0062.0030.00 95.8383.3375.0070.8360.4227.08 0.904(0.142-1.922)

Table 5 Insecticidal activity of Cry1Ah protein against first instar larvae of corn borer (Ostrina furnacalis) sample Concentrationug/g concentration test insect Insect number% just% LC _50ug /g CKCry1Ah 1851 144144144144 9144143139 6.25100.0099.3196.53 100.0099.2696.30 0.05012 (0.024-0.096) 0.10.010.005 144144144 934510 64.5831.256.94 62.2226.670.74

According to the results of the above tables, Cry1Ah has significant insecticidal activity against diamondback moth, cotton bollworm, Asian corn borer, and rice stem borer, and the LC ₅₀ values for the four tested pests are lower than several known highly toxic Force Bt insecticidal protein. The BtCry proteins with the highest toxicity to these four pests found internationally are:

diamondback moth, Cry1Ac (LC50=1.85 μg/ml); cotton bollworm, Cry1Ac (LC50=12.26 μg/ml;

Asian corn borer, Cry1Ac (LC50=0.134 μg/g); Rice stem borer Cry1Ab (LC50=1.81 μg/g).

It can be known that the toxicity of Cry1Ah of the present invention is stronger than these several known insecticidal proteins.

The above results are the insecticidal activity of the 134kDa component of the full-length Cry1Ah protein.

Studies on the Cry1Ah active region have shown that a polypeptide fragment (75kDa) (as shown in SEQ ID NO 4) of a total of 667 amino acids from the start codon ATG to 2001bp (as shown in SEQ ID NO 3) is also highly virulent, It is an essential segment for activity, and its poisonous effect on soybean borer, diamondback moth, cotton bollworm and rice stem borer is equivalent to that of the full-length Cry1Ah protein. This fragment can be completely used in the construction of insect-resistant transgenic plants.

Embodiment 8, cry1Ah full-length gene and 2.0kb fragment are compared with other Bt cry1 gene homology

The results of homologous comparison are shown in Tables 6, 7, 8, and 9 below:

Table 6 Homology comparison between cry1Ah full-length gene and other Bt cry1A genes 1Aa11Ag11Ab11Af11Ae11Ad11Ac11Ah cry1Aa1100 cry1Ag188100 cry1Ab18288100 cry1Af1677279100 cry1Ae181879075100 cry1Ad17884837289100 cry1Ac1827878637770100 cry1Ah71616751645984100

Table 7 Homology comparison between cry1Ah gene 2kb fragment and other Bt cry1A genes 1Ah 2kb1Ac 2kb1Ab 2kb1Af 2kb1Aa 2kb1Ag 2kb1Ad 2kb1Ae 2kb 1Ah 2kb100 1Ac 2kb84100 1Ab 2kb7288100 1Af 2kb718698100 1Aa 2kb66809290100 1Ag 2kb6478898895100 1Ad 2kb677787869288100 1Ae 2kb73849594898789100

Table 8 Homology comparison between Cry1Ah full-length protein and other BtCry1A proteins Cry1Aa1Cry1Ag1Cry1Ad1Cry1Ab1Cry1Af1Cry1Ae1Cry1Ac1Cry1Ah Cry1Aa1100 Cry1Ag194100 Cry1Ad19287100 Cry1Ab1908686100 Cry1Af167646375100 Cry1Ae19086899370100 Cry1Ac1848180866386100 Cry1Ah72697279567686100

Table 9 Homology comparison between Cry1Ah 75kDa polypeptide and other Bt Cry1A polypeptides 1Aa2k1Ag2k1Ad2k1Ab2k1Ac2k1Ah2k1Ae2k1Af2k 1Aa2k100 1Ag2k93100 1Ad2k9287100 1Ab2k908685100 1Ac2k76737284100 1Ah2k5957616782100 1Ae2k888486958267100 1Af2k888486958267100100

Homology comparison results show that the cry1Ah full-length gene and the 2kb fragment have the highest nucleotide homology with the cry1Ac full-length gene, reaching 84% (Table 6, 7); the amino acid homology between the Cry1Ah protein and the Cry1Ac protein is 86% (see Table 8); while the amino acid homology between the Cry1Ah74kDa polypeptide and the Cry1Ac protein is 82% (see Table 9), and the homology with other Cry1A proteins is lower than this value, for example, the homology with Cry1Ab is 67%, and the homology with Cry1Aa is 67%. The homology is 59%.

Figures 14 to 17 are the nucleotide sequences of the full-length cry1Ah gene and other cry1-like genes, the nucleotide sequences of the cry1Ah 2.0kb gene and other cry1-like genes, the full-length amino acid sequences of the Cry1Ah protein and other cry1-like proteins, Homologous phylogenetic tree of the N-terminal 667 amino acid sequences of Cry1Ah protein and other Cry1-like proteins. Whether it is from the analysis of nucleotide sequence or amino acid sequence, and whether it is full-length or half-length, cry1Ah (Cry1Ah) and cry1Ac (Cry1Ac), cry1Ab (Cry1Ab), cry1Ae (Cry1Ae), cry1Ad (Cry1Ad) and The genetic distance of cry1Af (Cry1Af) is relatively close; however, the biological activity is quite different.

Therefore, it can be known that if the cry1Ah half-length gene (2001kb, SEQ ID NO 3) is used as the material for the development of transgenic insect-resistant plants, it can more effectively overcome the current problems of high gene homology and single species, and at the same time it is expected to obtain Engineered plants with better pest resistance.

Attachment: DNA sequence and protein sequence involved in the present invention

SEQ ID NO 1 (nucleotide sequence of cry1Ah gene)

atgaaaaaca gtatcaaatt atcagaactt tggtatttca atgaaagaaa atggaggtat 60

tttatggaga tagtgaataa tcagaatcaa tgcgtgcctt ataattgttt gaataatccc 120

gaaatcgaaa tattagaagg cggaagaata tcagttggta ataccccaat tgatatttct 180

ctttcgctta ctcagtttct tttgagtgaa tttgtcccag gtgcggggtt tgtattagga 240

ttaattgatt taatatgggg atttgtaggt ccttcccaat gggacgcatt tcttgctcaa 300

gtggaacagt taattaacca aagaatagca gaagctgtaa gaaatacagc aattcaggaa 360

ttagagggaa tggcacgggt ttatagaacc tatgctactg cttttgctga gtgggaaaaa 420

gctcctgatg accccagagct aagagaagca ctacgtacac aatttacagc aactgagact 480

tatataagtg gaagaatatc cgttttaaaa attcaaactt ttgaagtaca gctgttatca 540

gtgtttgccc aagctgcaaa tttacattta tctttattaa gagacgttgt gttttttggg 600

caaagatggg gtttttcaac gacaaccgta aataattact acaatgattt aacagaaggg 660

attagtacct atacagatta tgctgtacgc tggtacaata cgggattaga acgtgtatgg 720

ggaccggatt ctagagattg ggtaaggtat aatcaattta gaagagaatt aacactaact 780

gtattagata tcgttgctct gttcccgaat tatgatagta gaagatatcc aattcgaaca 840

gtttcccaat taacaagaga aatttataca aacccagtat tagaaaattt tgatggtagt 900

tttcgaggct cggctcaggg catagaaaga agtattagga gtccacattt gatggatata 960

cttaacagta taaccatcta tacggatgct catagggggtt attattattg gtcagggcat 1020

caaataatgg cttctcctgt cggtttttcg gggccagaat tcacgtttcc gctatatgga 1080

accatgggaa atgcagctcc acaacaacgt attgttgctc aactaggtca gggcgtgtat 1140

agaacattat cctctacttt ttatagaaga ccttttaata tagggataaa taatcaacaa 1200

ctatctgttc ttgacgggac agaatttgct tatggaacct cctcaaattt gccatccgct 1260

gtatacagaa aaagcggaac ggtagattcg ctggatgaaa taccaccaca gaataacaac 1320

gtgccaccta ggcaaggatt tagtcatcga ttaagccatg tttcaatgtt tcgttcaggc 1380

tctagtagta gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1440

gaatttaata atataattgc atcggatagt attackcaaa tccctgcagt gaagggaaac 1500

tttcttttta atggttctgt aatttcagga ccaggatta ctggtgggga cttagttaga 1560

ttaaatagta gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1620

ccatcgacat ctaccagata tcgagttcgt gtacggtatg cttctgtaac cccgattcac 1680

ctcaacgtta attggggtaa ttcatccatt ttttccaata cagtaccagc tacagctacg 1740

tcattagata atctacaatc aagtgatttt ggttattttg aaagtgccaa tgcttttaca 1800

tcttcattag gtaatatagt aggtgttaga aattttagtg ggactgcagg agtgataata 1860

gacagatttg aatttattcc agttactgca acactcgagg ctgaatataa tctggaaaga 1920

gcgcagaagg cggtgaatgc gctgtttacg tctacaaacc aactagggct aaaaacaaat 1980

gtaacggatt atcatattga tcaagtgtcc aatttagtta cgtgtttatc ggatgaattt 2040

tgtctggatg aaaagcgaga attgtccgag aaagtcaaac atgcgaagcg actcagtgat 2100

gaacgcaatt tactccaaga ttcaaatttc aaagacatta ataggcaacc agaacgtggg 2160

tggggcggaa gtacagggat taccatccaa ggagtggatg acgtatttaa agaaaattac 2220

gtcacactat caggtacctt tgatgagtgc tatccaacat atttgtatca aaaaatcgat 2280

gaatcaaaat taaaagcctt tacccgttat caattaagag ggtacatcga agatagtcaa 2340

gatttagaag tttatttgat ccgttacaat gcaaaacacg aaacgttaaa cgtgccaggt 2400

acgggttcct tatggccact tgcagttaaa agtccaattg gaaggtgcgg tgaaccgaat 2460

cgatgtgcac atcattccca tcatttctcc ttggacattg atgtaggatg tacagactta 2520

aatgaggatt taggcgtatg ggtgatattc aagattaaga cacaagatgg ccatgcgaaa 2580

ataggaaatc tagaatttct cgaagagaag cttttattag gagaagcatt agcacgtgtg 2640

aagaaagcgg agaaaaaatg gagagacaaa cgcgaaaaat tggaatggga aacaaatatt 2700

gtttataaag aggcaaaaga atctgtagat gctttatcg tagattctca atataataga 2760

ttacaaacgg atacgaacat tgcgatgatt catgcggcag ataaacgcgt tcatcgaatc 2820

cgagaagcgt atttgccaga gttatctgtg attccgggtg tcaatgcggc tattttcgaa 2880

gaattagaag gtcttatttt caccgcattc tccctatatg atgcgagaaa tgtcattaaa 2940

aacggagatt tcaattatgg tttatcatgc tggaatgtga aagggcatgt agatgtagaa 3000

gaacaaaaca accaccgttc cgtccttgtt atcccagaat gggaagcaga agtgtcccaa 3060

gaagttcgtg tctgtccagg tcgtggctat atccttcgtg ttacagcgta caaagaggga 3120

tatggagagg gctgcgtaac gatccatgag atcgaagaca atacagacga actgaaattc 3180

agcaactgtg tagaagagga agtatatcca aacaacacgg taacgtgtaa tgattatact 3240

gcgactcaag aagaatatga gggtacgtac acttctcgta atcgaggata tgacggagcc 3300

tatgaaagca attcttctgt accagctgat tatgcatcag cctatgaaga aaaagcgtat 3360

acagatggac gaagagacaa tccttgtgaa tctaacagag gatataggga ttacacacca 3420

ctaccagctg gctatgtgac aaaagaatta gagtacttcc cagaaaccga taaggtatgg 3480

attgagatcg gagaaacgga aggaacattc attgtggata gcgtggaatt actccttatg 3540

gaggaatag 3549

SEQ ID NO 2 (protein sequence encoded by cry1Ah gene):

MKNSIKLSEL WYFNERKWRY FMEIVNNQNQ CVPYNCLNNP EIEILEGGRI SVGNTPIDIS 60

LSLTQFLLSE FVPGAGFVLG LIDLIWGFVG PSQWDAFLAQ VEQLINQRIA EAVRNTAIQE 120

LEGMARVYRT YATAFAEWEK APDDPELREA LRTQFTATET YISGRISVLK IQTFEVQLLS 180

VFAQAANLHL SLLRDVVFFG QRWGFSTTTV NNYYNDLTEG ISTYTDYAVR WYNTGLERVW 240

GPDSRDWVRY NQFRRELTLT VLDIVALFPN YDSRRYPIRT VSQLTREIYT NPVLENFDGS 300

FRGSAQGIER SIRSPHLMDI LNSITIYTDA HRGYYYWSGH QIMASPFGFS GPETFPLYG 360

TMGNAAPQQR IVAQLGQGVY RTLSSTFYRR PFNIGINNQQ LSVLDGTEFA YGTSSNLPSA 420

VYRKSGTVDS LDEIPPQNNN VPPRQGFSHR LSHVSMFRSG SSSSVSIIRA PMFSWIHRSA 480

EFNNIIASDS ITQIPAVKGN FLFNGSVISG PGFTGGDLVR LNSSGNNIQN RGYIEVPIHF 540

PSTSTRYRVR VRYASVTPIH LNVNWGNSSI FSNTVPATAT SLDNLQSSDF GYFESANAFT 600

SSLGNIVGVR NFSGTAGVII DRFEFIPVTA TLEAEYNLER AQKAVNALFT STNQLGLKTN 660

VTDYHIDQVS NLVTCLSDEF CLDEKRELSE KVKHAKRLSD ERNLLQDSNF KDINRQPERG 720

WGGSTGITIQ GVDDVFKENY VTLSGTFDEC YPTYLYQKID ESKLKAFTRY QLRGYIEDSQ 780

DLEVYLIRYN AKHETLNVPG TGSLWPLAVK SPIGRCGEPN RCAHHSHHFS LDIDVGCTDL 840

NEDLGVWVIF KIKTQDGHAK IGNLEFLEEK LLLGEALARV KKAEKKWRDK REKLEWETNI 900

VYKEAKESVD ALFVDSQYNR LQTDTNIAMI HAADKRVHRI REAYLPELSV IPGVNAAIFE 960

ELEGLIFTAF SLYDARNVIK NGDFNYGLSC WNVKGHVDVE EQNNHRSVLV IPEWEAEVSQ 1020

EVVRVCPGRGY ILRVTAYKEG YGEGCVTIHE IEDNTDELKF SNCVEEEVYP NNTVTCNDYT 1080

ATQEEYEGTY TSRNRGYDGA YESNSSVPAD YASAYEEKAY TDGRRDNPCE SNRGYRDYTP 1140

LPAGYVTKEL EYFPETDKVW IEIGETEGTF IVDSVELLLM EE 1182

SEQ ID NO 3 (cry1Ah2k gene nucleotide sequence)

atgaaaaaca gtatcaaatt atcagaactt tggtatttca atgaaagaaa atggaggtat 60

tttatggaga tagtgaataa tcagaatcaa tgcgtgcctt ataattgttt gaataatccc 120

gaaatcgaaa tattagaagg cggaagaata tcagttggta ataccccaat tgatatttct 180

ctttcgctta ctcagtttct tttgagtgaa tttgtcccag gtgcggggtt tgtattagga 240

ttaattgatt taatatgggg atttgtaggt ccttcccaat gggacgcatt tcttgctcaa 300

gtggaacagt taattaacca aagaatagca gaagctgtaa gaaatacagc aattcaggaa 360

ttagagggaa tggcacgggt ttatagaacc tatgctactg cttttgctga gtgggaaaaa 420

gctcctgatg accccagagct aagagaagca ctacgtacac aatttacagc aactgagact 480

tatataagtg gaagaatatc cgttttaaaa attcaaactt ttgaagtaca gctgttatca 540

gtgtttgccc aagctgcaaa tttacattta tctttattaa gagacgttgt gttttttggg 600

caaagatggg gtttttcaac gacaaccgta aataattact acaatgattt aacagaaggg 660

attagtacct atacagatta tgctgtacgc tggtacaata cgggattaga acgtgtatgg 720

ggaccggatt ctagagattg ggtaaggtat aatcaattta gaagagaatt aacactaact 780

gtattagata tcgttgctct gttcccgaat tatgatagta gaagatatcc aattcgaaca 840

gtttcccaat taacaagaga aatttataca aacccagtat tagaaaattt tgatggtagt 900

tttcgaggct cggctcaggg catagaaaga agtattagga gtccacattt gatggatata 960

cttaacagta taaccatcta tacggatgct catagggggtt attattattg gtcagggcat 1020

caaataatgg cttctcctgt cggtttttcg gggccagaat tcacgtttcc gctatatgga 1080

accatgggaa atgcagctcc acaacaacgt attgttgctc aactaggtca gggcgtgtat 1140

agaacattat cctctacttt ttatagaaga ccttttaata tagggataaa taatcaacaa 1200

ctatctgttc ttgacgggac agaatttgct tatggaacct cctcaaattt gccatccgct 1260

gtatacagaa aaagcggaac ggtagattcg ctggatgaaa taccaccaca gaataacaac 1320

gtgccaccta ggcaaggatt tagtcatcga ttaagccatg tttcaatgtt tcgttcaggc 1380

tctagtagta gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1440

gaatttaata atataattgc atcggatagt attackcaaa tccctgcagt gaagggaaac 1500

tttcttttta atggttctgt aatttcagga ccaggatta ctggtgggga cttagttaga 1560

ttaaatagta gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1620

ccatcgacat ctaccagata tcgagttcgt gtacggtatg cttctgtaac cccgattcac 1680

ctcaacgtta attggggtaa ttcatccatt ttttccaata cagtaccagc tacagctacg 1740

tcattagata atctacaatc aagtgatttt ggttattttg aaagtgccaa tgcttttaca 1800

tcttcattag gtaatatagt aggtgttaga aattttagtg ggactgcagg agtgataata 1860

gacagatttg aatttattcc agttactgca acactcgagg ctgaatataa tctggaaaga 1920

gcgcagaagg cggtgaatgc gctgtttacg tctacaaacc aactagggct aaaaacaaat 1980

gtaacggatt atcatattga t 2001

SEQ ID NO 4 (amino acid sequence of protein encoded by cry1Ah2kb gene)

MKNSIKLSEL WYFNERKWRY FMEIVNNQNQ CVPYNCLNNP EIEILEGGRI SVGNTPIDIS 60

LSLTQFLLSE FVPGAGFVLG LIDLIWGFVG PSQWDAFLAQ VEQLINQRIA EAVRNTAIQE 120

LEGMARVYRT YATAFAEWEK APDDPELREA LRTQFTATET YISGRISVLK IQTFEVQLLS 180

VFAQAANLHL SLLRDVVFFG QRWGFSTTTV NNYYNDLTEG ISTYTDYAVR WYNTGLERVW 240

GPDSRDWVRY NQFRRELTLT VLDIVALFPN YDSRRYPIRT VSQLTREIYT NPVLENFDGS 300

FRGSAQGIER SIRSPHLMDI LNSITIYTDA HRGYYYWSGH QIMASPFGFS GPETFPLYG 360

TMGNAAPQQR IVAQLGQGVY RTLSSTFYRR PFNIGINNQQ LSVLDGTEFA YGTSSNLPSA 420

VYRKSGTVDS LDEIPPQNNN VPPRQGFSHR LSHVSMFRSG SSSSVSIIRA PMFSWIHRSA 480

EFNNIIASDS ITQIPAVKGN FLFNGSVISG PGFTGGDLVR LNSSGNNIQN RGYIEVPIHF 540

PSTSTRYRVR VRYASVTPIH LNVNWGNSSI FSNTVPATAT SLDNLQSSDF GYFESANAFT 600

SSLGNIVGVR NFSGTAGVII DRFEFIPVTA TLEAEYNLER AQKAVNALFT STNQLGLKTN 660

VTDYHID 667

Claims (4)

1. one kind has toxic bacillus thuringiensis crylAh gene to insect, and the nucleotide sequence that it is characterized in that described gene is shown in SEQ ID NO 1.

2. one kind has toxic protein to insect, it is characterized in that this protein is the coded by said gene by claim 1, and this proteinic aminoacid sequence is shown in SEQ ID NO 2.

3. the application of the described protein of claim 2 in killing lepidoptera pest.

4. the purposes of the described crylAh gene of claim 1 in transforming microorganism or plant.

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