CN114032247A - Application of insecticidal gene cry2Ah-vp and cry9Ee combination in insect-resistant plants - Google Patents

Abstract

The invention relates to application of a combination of insecticidal genes cry2Ah-vp and cry9Ee in insect-resistant plants. The invention transforms the cry2Ah-vp and cry9Ee genes with high toxicity to lepidoptera pests and synthesizes a new DNA sequence, the two genes are respectively transferred into corn by utilizing an agrobacterium-mediated method, and 2HVB5 and 9EG1 transformation events with high resistance to armyworm and corn borer are obtained by screening. The cry2Ah-vp and cry9Ee genes are combined by a gene polymerization method to obtain 2HVB5/9EG1 transgenic corn material. The experimental result shows that the transgenic corn combining the cry2Ah-vp and cry9Ee genes has high insecticidal activity on armyworm and corn borer, especially resistant corn borer. The invention has very important significance for the cooperative expression culture of insect-resistant plants, pest control and resistance control by transforming plants with cry2Ah-vp and cry9Ee genes.

Description

Application of insecticidal gene cry2Ah-vp and cry9Ee combination in insect-resistant plants

Technical Field

The invention belongs to the technical field of biological control, and particularly relates to application of a combination of insecticidal genes cry2Ah-vp and cry9Ee in insect-resistant plants.

Background

Insect damage is an important factor for crop yield reduction in the world, so that about 10% of the total yield of grains is lost every year, and the direct economic loss is up to hundreds of billions of dollars. The prevention and treatment in the past decades mainly depends on chemical pesticides, which make great contribution to agricultural production and cause serious consequences such as environmental pollution, poisoning of people and livestock, ecological imbalance and the like. The development and application of transgenic technology provides a safe and effective new method for crop pest resistance.

Bacillus thuringiensis (abbreviated as Bt) is a gram-positive bacterium with wide distribution. It produces protein-like parasporal crystals (parasporal crystals) with specific insecticidal activity against a variety of insects, as well as nematodes, mites and protozoa, while sporulating (Schnepf E.et al,1998, Microbiology and Molecular Biology Review,62(3): 775. 806). This Insecticidal Crystal Protein (ICPs), also known as Delta-endotoxin, first dissolves in the insect midgut to become protoxin and then is degraded by gut proteases to a toxin with specific activity that binds to a specific receptor on the midgut (Craig,2007, Microbiology and Molecular Biology Review,71(2):255 and 281), leading to insect death. The Bacillus thuringiensis insecticidal crystal protein has good insecticidal effect, is harmless to human and livestock, does not pollute the environment, and is widely applied to biological control of pests.

Since the commercialization of transgenic crops in 1996, the use of transgenic crops has increased rapidly, 1.904 million hectares of transgenic crops have been grown in 29 countries worldwide in 2019, and the growing area of transgenic crops has increased by about 112 times compared to 1996 (isama Briefs, 2019). However, the insect-resistant gene types of the current commercial transgenic insect-resistant crops are single, and mainly belong to cry1A types. The long-time large-area popularization and planting has the risks of reduction of refuge of pests and increase of pesticide resistance of the pests. Therefore, there is a need to use new highly toxic insecticidal genes or new genes to avoid the risk of increased resistance of pests.

The Cry2Ah gene is obtained by mixing and collecting soil samples and exempting from culture, and the Cry2Ah protein has weight suppression activity on corn borers, growth suppression activity on cotton bollworms and good growth suppression activity on Cry1Ac resistant cotton bollworms (Shu C L, Zhang J T, Chen Gui H, Liang G M, He K L, Crickmore N, Huang D F, Zhang J, Song F P.2013, Journal of Invertebrate Pathology 114: 31-33). The mutant Cry2Ah-vp is obtained by inserting a proline after valine at position 354 of Cry2 Ah. After Cry2Ah and Cry2Ah-vp genes were transferred into tobacco plants, respectively, experiments on insecticidal activity against cotton bollworm and Cry1Ac resistant cotton bollworm were performed, and the results showed that Cry2Ah-vp had higher insecticidal activity than Cry2Ah (Li S Y, Wang Z Y, Zhou Y, Li C H, Wang G P, Wang H, Zhang J, Liang G M, Lang Z h.2018, Scientific Reports,8(1): 508). The applicant describes in detail a cry2Ah-vp gene sequence, a vector and application thereof in a patent of artificially synthesized insecticidal gene with high toxicity to lepidoptera pests and application thereof (patent number: ZL201710145166.6) and a patent of application of cry2Ah-vp gene in anti-myxoma (patent number: ZL201910191397. X).

The Cry9Ee gene is cloned from Bacillus thuringiensis T03B001 strain, Cry9Ee and Cry1A have no cross resistance, and have high toxicity to diamondback moth, corn borer and Cry1Ac resistant diamondback moth (Shu C L, Su H Q, Zhang J, He K L, Huang D F, Song F P.2013, Appl Microbiol Biotechnol,97: 9705-.

The monovalent Bt insecticidal gene has the problems of limited toxicity, narrow insecticidal spectrum and resistance generation of pests in application, and researchers use the combined application of two or more Bt insecticidal genes to achieve the effects of expanding the insecticidal spectrum and controlling the resistance. However, different Bt gene combinations can produce different synergistic and antagonistic results due to different action receptors, and the Zhangjie research group finds that Bt proteins Cry1Ai and Cry9Aa, Cry1Ea and Cry9Aa have synergistic effect, while the combination of Cry1Ai and Cry1Ea has no obvious synergistic effect (Chuaigiline, Balinglong dragon, Songfiping, yellow Bob, Zhangjie.2013, plant protection, 39(1): 66-70); yang et al found that Vip3Aa16 in combination with Cry1 had a combined synergistic effect on the insecticidal activity of Oriental myxoworms, whereas Vip3Ca in combination with Cry1 and Cry2 did not (Yang J, Quan Y, Sivaprasath P, Shabbir M, Wang Z, Ferre J, He K.2018, Toxins,10: 454). The selection of gene combinations with synergistic effects is a new gene source for creating a new generation of insect-resistant plants.

Disclosure of Invention

Aiming at the requirements in the field, the invention provides the application of two gene combinations of Cry2Ah-vp and Cry9Ee, which are insecticidal proteins with high toxicity to lepidoptera, in the prevention and treatment of lepidoptera pests, the genes of Cry2Ah-vp and Cry9Ee are subjected to codon optimization and are synthesized into new DNA sequences, a plant expression vector is constructed to transform maize, the genes of Cry2Ah-vp and Cry9Ee are combined together by a gene polymerization method, and experiments show that the synergistic expression of the genes of Cry2Ah-vp and Cry9Ee endows the maize with the effects of high resistance to armyworm, ostrinia nubilalis and Cry1A resistant ostrinia nubilalis, and the invention has application prospects in pest resistance treatment.

The nucleotide sequence of the cry2Ah-vp gene is shown in SEQ ID NO. 3.

The plant expression vector is pC2HBvp, and the skeleton vector is pCAMBIA 3300.

The structure of the plant expression vector pC2HBvp is shown in figure 1.

The nucleotide sequence of the cry9Ee gene is shown in SEQ ID NO. 4.

The plant expression vector is pC9EG, and the skeleton vector is pCAMBIA 2300.

The structure of the plant expression vector pC9EG is shown in figure 2.

The Cry2Ah-vp and Cry9Ee protein combination is applied to insect-resistant plants, wherein the amino acid sequence of the Cry2Ah-vp protein is shown as SEQ ID NO.5, and the amino acid sequence of the Cry9Ee protein is shown as SEQ ID NO. 6.

The application is to transform the expression vector containing Cry2Ah-vp gene and Cry9Ee gene into plants to make the plants express Cry2Ah-vp and Cry9Ee proteins, so that the transformed plants have the characteristic of insect resistance.

In order to transform different plants of the expression vectors respectively containing Cry2Ah-vp gene and Cry9Ee gene, Cry2Ah-vp and Cry9Ee proteins are respectively expressed by the different plants, and then Cry2Ah-vp and Cry9Ee genes are polymerized into the same plant by a hybridization method, so that the hybrid plants express Cry2Ah-vp and Cry9Ee proteins, thereby enabling the hybrid plants to have insect-resistant property.

The nucleotide sequence of the cry2Ah-vp gene in the expression vector containing the cry2Ah-vp gene is shown in SEQ ID NO.3, and the expression vector containing the cry9Ee gene is the vector.

The expression vector plant containing cry2Ah-vp gene is pC2HBvp, and the backbone vector is pCAMBIA 3300.

The insect-resistant is lepidopteran pest-resistant.

The transformation method is an agrobacterium-mediated method, and the plant is corn, but is not limited to corn.

The invention carries out codon optimization and mutation of nucleotide sequence of cry2Ah (SEQ ID NO.1) gene, carries out codon optimization on the nucleotide sequence of cry9Ee (SEQ ID NO.2), synthesizes new genes cry2Ah-vp (SEQ ID NO.3) and cry9Ee (SEQ ID NO.4) in an artificial synthesis mode, respectively constructs plant expression vectors pC2HBvp and pC9EG containing cry2Ah-vp and cry9Ee, transfers the plants into corn by an agrobacterium-mediated method, and respectively screens and obtains transgenic cry2Ah-vp gene corn event 2HVB5 and transgenic cry9Ee gene corn event 9EG1 through PCR identification and insect resistance experiments. Two transgenic events are used for polymerizing cry2Ah-vp and cry9Ee genes into the same corn plant through a hybridization method, and PCR detection results show that the cry2Ah-vp and cry9Ee genes are integrated into a corn genome. The insect resistance experiment result shows that the transgenic corn with the combination of Cry2Ah-vp and Cry9Ee genes has good insecticidal activity on armyworm and corn borer, particularly Cry1Ab resistant corn borer and Cry1Ac resistant corn borer, and the armyworm, the corn borer, the Cry1Ab resistant corn borer and the Cry1Ac resistant corn borer all die after being inoculated with insects for 3 days.

The combined application of the cry2Ah-vp and cry9Ee genes has good insecticidal activity on armyworm, corn borer and resistant corn borer, which is helpful for reducing the risk of pest resistance increase, and the transgenic corn applied in combination is a germplasm resource for corn pest-resistant breeding and pest control, and has good commercialization prospect in corn pest-resistant breeding.

Drawings

FIG. 1 is a schematic diagram of the construction of plant expression vector pC2 HBvp.

FIG. 2 is a schematic diagram of the construction of plant expression vector pC9 EG.

FIG. 3 PCR detection of cry2Ah-vp gene of pC2 HBvp-transferred vector maize

Wherein M is DNA molecular weight standard Super Marker; CK +: using plasmid pC2HBvp as template amplification product; CK-: using non-transgenic corn genome DNA as a template amplification product; 0: blank, using water as template amplification product; 1-5: part of the maize genome DNA transformed with cry2Ah-vp gene is used as a template amplification product.

FIG. 4 PCR detection of maize cry9Ee gene transformed with pC9EG vector

Wherein M is DNA molecular weight standard Super Marker; CK +: using plasmid pC9EG as a template to amplify the product; CK-: using non-transgenic corn genome DNA as a template amplification product; 0: blank, using water as template amplification product; 1-5: part of the cry9Ee transgenic corn genome DNA is used as a template amplification product.

FIG. 5 PCR detection of transgenic maize with cry2Ah-vp and cry9Ee gene combinations.

Wherein the cry2Ah-vp gene (A), the bar gene (B), the cry9Ee gene (C), and the GAT gene (D);

wherein M is DNA molecular weight standard Super Marker; CK +: using plasmid pC2HBvp or pC9EG as template amplification product; CK-: using non-transgenic corn genome DNA as a template amplification product; 0: blank, using water as template amplification product; 1-3: transgenic corn genomic DNA of the combination of cry2Ah-vp and cry9Ee genes is used as a template amplification product.

Detailed Description

The present invention will be described in further detail with reference to examples.

The following biological materials are stored in the laboratory of the applicant and can be dispensed to the outside.

1. Codon modification and optimization of Bt cry2Ah-vp and cry9Ee genes

Bt cry2Ah (GenBank No. ACL13555.1) is a gene cloned from Bt strain isolated in China, the coding region of the gene has 1899bp, the nucleic acid sequence is shown in SEQ ID NO.1, and the coded protein consists of 632 amino acids. The Cry2Ah-vp mutant protein is characterized in that a proline (Pro) is added after a valine (Val) at position 354 of the Cry2Ah1 protein, a 3-base CCC is added after a 1062bp position on a nucleotide sequence, proline is encoded, and the optimized Cry2Ah-vp nucleotide sequence is shown in SEQ ID NO.3, and an amino acid sequence is shown in SEQ ID NO. 5.

Bt cry9Ee (GenBank No. ADE60738.1) is cloned from Bacillus thuringiensis T03B001 strain, the coding region of the gene has 3471bp, the nucleic acid sequence is shown in SEQ ID NO.2, and the coded protein consists of 1156 amino acids. Through analysis of the Cry9Ee protein sequence domain, there are three conserved domains: the amino acids 86-293 as domain I (endo _ N), 302-508 as domain II (endo _ M) and 510-657 as domain III (endo _ C) are essential active segments. In the present invention, the coding sequence (2202bp) of amino acids 1-734 of Bt cry9Ee gene was selected and optimized according to the preferred codons of plants. The GC content of the original cry9Ee gene (2202bp) is 39.2 percent, the GC content of the modified gene is 63.5 percent, the similarity with the original cry9Ee nucleic acid sequence is 66.53 percent, the optimized cry9Ee nucleic acid sequence is shown in SEQ ID NO.4, and the amino acid sequence is shown in SEQ ID NO. 6.

2. Construction of plant expression vectors

The plant expression vector pC2HBvp containing cry2Ah-vp gene comprises a maize Ubiquitin promoter, an optimized cry2Ah-vp gene and an NOS terminator, and the construction process is explained in detail in the patent of application of cry2Ah-vp gene in anti-myxoworm (patent number: ZL201910191397.X), the schematic diagram of the pC2HBvp vector is shown in figure 1, and the vector contains bar gene and cry2Ah-vp gene.

The optimized cry9Ee gene is artificially synthesized, and an OMK sequence for enhancing gene expression is added at the upstream of the cry9Ee gene. pUC57-UN is an intermediate vector containing a Ubiquitin promoter and a NOS terminator (the plasmid was stored in the encyclopedia of institute of Biotechnology, national academy of agricultural sciences, available to the public), and the synthesized OMK-cry9Ee fragment was ligated into pUC57-UN vector by BamHI and KpnI digestion, and the cry9Ee expression cassette was ligated into pCGAT vector by HindIII and EcoRI digestion. The pCGAT intermediate vector is a vector obtained by adding an OMK sequence for enhancing gene expression to the upstream of a artificially synthesized and optimized glyphosate acetylase GAT gene, digesting an nptII gene in T-DNA by XhoI in a commercial vector pCAMBIA2300, and inserting a synthesized OMK-GAT fragment into the pCAMBIA2300 in a seamless cloning manner (the plasmid is stored in a Langerhans Macro subject group of the institute of biotechnology of Chinese academy of agricultural sciences and can be provided for the public). The expression cassette containing cry9Ee gene is connected with pCGAT to construct the final vector pC9EG, the schematic diagram of the vector is shown in FIG. 2, and the vector contains glyphosate acetyltransferase gene GAT and insecticidal gene cry9 Ee.

3. Acquisition of cry2Ah-vp and cry9Ee transgenic maize

The vectors pC2HBvp and pC9EG are respectively transformed into Agrobacterium EHA105 by a freeze-thaw method, and PCR is carried out for identification. Taking freshly stripped young maize embryos of about 1.2mm as a material, placing the young embryos in an infection culture medium for one hour, washing the young embryos with the infection culture medium once, immersing the young embryos in 100 mu M of acetosyringone added agrobacterium liquid, and placing for 5 minutes. Taking out, drying with sterilized filter paper, placing on co-culture medium, co-culturing at 26 deg.C in dark for 3 days, and setting reference. Transferring the young embryos to a recovery culture medium for culturing for 10 days until callus is induced, transferring the callus to a screening culture medium containing a corresponding screening agent after bud removal, carrying out subculture once every two weeks, transferring the resistant callus to a regeneration culture medium through screening for 6 weeks, carrying out visible light differentiation, starting to generate green bud spots after the callus is exposed for about one week, splitting the callus blocks to separate the green bud spots, transferring the green bud spots to the regeneration culture medium for culturing, facilitating the growth of main stems, transferring the main stems to the regeneration culture medium for inducing rooting when the main stems are elongated to 3-4 cm, and transferring the corn plants to a small greenhouse flowerpot for growing after the corn plants are strong and have developed root systems. After the cultivation is continued for two weeks, the transformed seedlings are transferred to a greenhouse after the growth state is good, the silks are covered by paper bags after the silks of the female ears, pollination is carried out after pollen of the male ears is scattered, and the fruits are harvested. The transgenic pC2HBvp vector plant 31 and the transgenic pC9EG vector plant 91 were obtained by maize transformation. Transformed plants were verified by PCR and the primer sequences are shown in Table 1. In 20. mu.L of the reaction mixture containing 100ng of the DNA template,primers 0.1. mu.M each, 10. mu.L of 2 × taq MasterMix (Dye), ddH₂Make up to 20. mu.L of O. The cry2Ah-vp reaction conditions are: 94 ℃ for 5min, (94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 60s) for 30 cycles, 72 ℃ for 5 min. The cry9Ee reaction conditions were: 94 ℃ for 5min, (94 ℃ for 30s, 56.9 ℃ for 30s, 72 ℃ for 30s) for 30 cycles, 72 ℃ for 5 min. The PCR detection result of the cry2Ah-vp transgenic corn is shown in figure 3, and the PCR detection result of the cry9Ee transgenic corn is shown in figure 4. The results showed that the foreign gene cry2Ah-vp or cry9Ee gene had been introduced into the maize genome.

The culture medium comprises:

infection culture solution: n6 salt and N6 vitamin (Chu et al, Science Sinica, 1975, 18:659-668), 1.5 mg/L2, 4-D, 0.7/L g proline, 68.4g/L sucrose, 36g/L glucose (pH 5.2), filter sterilized and stored at 4 ℃; adding filtered and sterilized Acetosyringone (AS) before use, and the final concentration is 100 mu M;

co-culture medium: n6 salt and N6 vitamin, 1.5 mg/L2, 4-D, 0.7g/L proline, 30g/L sucrose, 3g/L plant gel (pH5.8), autoclaved, added with filtered sterilized silver nitrate to a final concentration of 0.85mg/L, 100. mu.M AS, 300mg/L cysteine;

recovering the culture medium: n6 salt and N6 vitamin, 1.5 mg/L2, 4-D, 0.7g/L proline, 30g/L sucrose, 0.5g/L MES, 4g/L plant gel (pH5.8), autoclaved and then added with 0.85mg/L silver nitrate and 200mg/L carbenicillin, filter sterilized;

screening a culture medium: adding 1mM glyphosate as a screening agent into a recovery culture medium;

regeneration culture medium: MS salt and MS vitamins, 30g/L sucrose, 100mg/L inositol, 3g/L vegetable gel (pH5.8), autoclaving.

The young maize embryos are freshly stripped young embryos 1.2mm long.

100 mu M acetosyringone is added into the agrobacterium liquid.

TABLE 1 primer sequences for PCR

4. Screening of cry2Ah-vp and cry9Ee transgenic insect-resistant maize

After the obtained cry2Ah-vp and cry9Ee transgenic corn materials are sown and the corn grows to the six-leaf stage, indoor bioassay is carried out. And (3) determining the insecticidal activity of the transgenic corn plant on myxozoa and sensitive corn borer larvae. Indoor bioassay of myxozoon and sensitive corn borer larvae uses 24-hole culture dish, and 1cm of culture dish is taken²The corn leaves with the same size are placed in 24 holes, one newly hatched larva is placed in each hole, and the number of live insects and dead insects is recorded every day. Three biological replicates of each treatment were performed, with leaves of Zheng 58 material from the inbred line as a control. Screening to obtain transgenic cry2Ah-vp corn material 2HVB5 and transgenic cry9Ee corn material 9EG1 with good insect resistance. The insect test result shows that the armyworm corrected mortality rate of the 2HVB5 material is 100% 4 days after the inoculation, and the corn borer corrected mortality rate of the 2HVB5 material is 100% 5 days after the inoculation; 4 days after inoculation, all the corn borers with 9EG1 material die; the insecticidal activity of the 9EG1 material on the armyworms is lower than that of the 2HVB5 material, the corrected mortality rate of the armyworms after the armyworms are inoculated for 5 days is 53.56%, and the individual test insects still survive after the armyworms are inoculated for 6 days (Table 2). Indoor biological activity identification shows that 2HVB5 has good insecticidal activity on armyworm and corn borer, and 9EG1 material has good insecticidal activity on corn borer.

TABLE 22 corrected mortality statistics for corn borer and armyworm for HVB5 and 9EG1

5. Hybridization combinations and assays for 2HVB5 and 9EG1

The Bt cry2Ah-vp gene and the cry9Ee gene belong to cry2 genes and cry9 genes, and the amino acid sequence similarity is 9.07 percent. The transgenic corn 2HVB5 and 9EG1 are combined by a hybridization method to obtain a 2HVB5/9EG1 combined material containing two genes of cry2Ah-vp and cry9 Ee. PCR detection of 2HVB5/9EG1 material was performed with primers specific to cry2Ah-vp, bar, cry9Ee and GAT genes, respectively, and the primer sequences are shown in Table 1. mu.L of the reaction mixture containing 100ng of DNA template, 0.1. mu.M each of primers, 10. mu.L of 2 XTaq MasterMix (Dye))，ddH₂Make up to 20. mu.L of O. The cry2Ah-vp reaction conditions are: 94 ℃ for 5min, (94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 60s) for 30 cycles, 72 ℃ for 5 min. The bar reaction conditions are as follows: 94 ℃ for 5min, (94 ℃ for 30s, 56 ℃ for 30s, 72 ℃ for 20s) for 30 cycles, 72 ℃ for 5 min. The cry9Ee reaction conditions were: 94 ℃ for 5min, (94 ℃ for 30s, 56.9 ℃ for 30s, 72 ℃ for 30s) for 30 cycles, 72 ℃ for 5 min. The GAT reaction conditions are as follows: 94 ℃ for 5min, (94 ℃ for 30s, 58.5 ℃ for 30s, 72 ℃ for 20s) for 30 cycles, 72 ℃ for 5 min. The PCR results are shown in FIG. 5. The results showed that the cry2Ah-vp, bar, cry9Ee and GAT genes in 2HVB5/9EG1 material could all be detected, indicating that the foreign gene had been integrated into the same material.

Indoor bioassay is carried out on 2HVB5/9EG1 materials containing 4 genes detected by PCR, and the insecticidal activity of the 2HVB5/9EG1 materials combined by Cry2Ah-vp and Cry9Ee genes on larvae of myxozoa, corn borers, Cry1Ab resistant corn borers and Cry1Ac resistant corn borers is detected. The insect test results are shown in Table 3, after 3 days of inoculation, the corrected mortality rates of the armyworm, the corn borer, the Cry1Ab resistant corn borer and the Cry1Ac resistant corn borer which are made of 2HVB5/9EG1 materials are all 100%, and the results show that the gene combination of Cry2Ah-vp and Cry9Ee has high insecticidal activity on the armyworm, the corn borer, the Cry1Ab resistant corn borer and the Cry1Ac resistant corn borer. Although 2HVB5 containing cry2Ah-vp gene alone and 9EG1 containing cry9Ee gene alone have good insecticidal activity on corn borers, still one individual insect survives 3 days after inoculation, and all test insects die 4 days or 5 days later. The results of statistical analysis show that the three materials of 2HVB5/9EG1, 2HVB5 and 9EG1 have extremely significant differences in the corrected mortality rate of corn borer (2HVB5/9EG1>9EG1>2HVB 5). The 2HVB5 material has good insecticidal activity on the armyworms, the corrected mortality rate of the armyworms after 3 days of inoculation is 94.38%, the insecticidal activity of the 9EG1 material on the armyworms is general, the corrected mortality rate of the armyworms after 3 days of inoculation is 39.49%, and the statistical analysis result shows that the corrected mortality rates of the three materials of 2HVB5/9EG1, 2HVB5 and 9EG1 on the armyworms are very different (2HVB5/9EG1>2HVB5>9EG 1). The 2HVB5 and 9EG1 materials have good insecticidal activity on Cry1Ab and Cry1Ac resistant corn borers, and statistical analysis results show that the corrected mortality rate of the 2HVB5/9EG1 on the Cry1Ab resistant corn borers is obviously different from that of the 9EG1 material, is greatly obviously different from that of the 2HVB5 material, and the 9EG1 material is not obviously different from that of the 2HVB5 material; the corrected mortality rate of the 2HVB5/9EG1 to Cry1Ac resistant corn borers is not significantly different from that of 9EG1 material, is very significantly different from that of 2HVB5 material, and is significantly different from that of 9EG1 and 2HVB5 material.

TABLE 32 HVB5/9EG1 Material corrected mortality statistics for myxozoa, Zea mays borer and resistant Zea mays borer

Note: capital letters indicate significant level differences (P <0.05), lowercase letters indicate extremely significant level differences (P <0.01)

The experimental result shows that the gene combination application of cry2Ah-vp and cry9Ee has high insecticidal activity on armyworm and corn borer, especially resistant corn borer, and is superior to a single gene. The cry2Ah-vp and cry9Ee gene combination can be used for breeding insect-resistant corn, has very important significance for preventing and controlling corn pests and controlling resistance, and has good industrialization prospect.

Sequence listing

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aatcagttat ggccaactaa tgataatgca gtatgggaag cttttatgga acaaatagaa 360

gggattatcg ctcaaagaat atcggagcaa gtagtaagga atgcgcttga tgccttaact 420

ggaatacacg attattatga ggaatattta gcggcattag aggagtggct ggaaagaccg 480

agcggcgcaa gggctaactt agcttttcag aggtttgaaa atctacatca attatttgta 540

agtcagatgc caagttttgg tagtggtcct ggtagtgaaa gagatgcggt agcattgctg 600

acagtatatg cacaagcagc gaatctccat ttgttgttat taaaagatgc agaaatttat 660

ggggcgagat ggggacttca acaaggccaa attaatttat attttaatgc tcaacaagat 720

cgcactcgaa tttataccaa tcattgtgtg gcaacatata atagaggatt aggagactta 780

agaggcacaa atactgaaag ttggttaaat taccatcaat tccgtagaga gatgacatta 840

atggcaatgg atttagtggc attattccca tactataatt tacgacaata tccaaacggg 900

gcaaaccctc agcttacacg tgatgtatat acagatccga ttgtatttaa tccatcagct 960

aatgtaggat tatgtagacg ttggggcaat aacccatata atacattttc ggaacttgaa 1020

aatgccttca ttcgcccgcc acattttttt gataggttga atagtttaac aattagtaga 1080

aatagatttg acgttggatc aaactttata gagccttggt ctggacatac gttacgccgt 1140

agttttctga acacttcggc agtacaagaa gatagttatg gccaaattac taatcaaaga 1200

acaacaatta atctaccagc taatggaact gggcgagtgg agtcaacagc agtagatttt 1260

cgtagcgcgc ttgtggggat atacggcgtt aatagagctt cttttattcc cggtggtgtg 1320

tttaatggca cgactcaacc ttctactgga ggatgtagag atttgtatga ttcaagtgat 1380

gaattaccac cagaagaaag tagtggaacg tttgaacata ggttatctca tgttaccttt 1440

ttaagtttta caactaatca ggctggatcc atagccaatg cagggcgcgt ccctacttat 1500

gtctggaccc atcgagatgt ggaccttaat aacacgatta ctgcagatag aattacacac 1560

ttaccattga taaaatcaaa tgtgcaacgc agtggtcgcg cagtaaaagg accaggattt 1620

acaggaggag atgtactccg aatgtcatca agtgatgctg atatatcaat aataggaata 1680

acggcaggtg caccgctaac acaacaatat cgtataagat tgcgttatgc ttcaaatgta 1740

gatgttacta tccgtttagt gagacaggac acccaaagta atataggaag cataaactta 1800

ttacgtacaa tgaacagtgg agaggagtca aggtatgaat catatcgtac tgtagagatg 1860

cctggtaatt ttagaatgac tagtagttca gcacagattc gactatttac tcaaggactt 1920

cgagtgaatg gagaattgtt tcttgatagt cttgaattta tcccagttaa tccgacacgt 1980

gaggcggaag aggatttaga agcagcgaag aaagcggtga cgagcttgtt tacacgtaca 2040

agtgatggat tacagataaa tgtgacagat taccaagtcg atcaggcggc aaatttagtg 2100

tcgtgcttat cagatgaaca atatgggcat gataaaaaga tgttattgga agccgtacgc 2160

gcagcaaaac gcctcagccg cgaacgcaac ttacttcaag atccagattt taatacaatc 2220

aatagtacag aagaaaatgg ctggaaggca agtaacggtg ttactattag cgagggcggt 2280

ccattcttta aaggtcgtgc acttcagtta gcaagcgcaa gagaaaatta tccaacatac 2340

atttatcaaa aagtagatgc atcggtgtta aagccttata cacgctatag actagatgga 2400

tttgtgaaga gtagtcaaga tttagaaatt gatctcatcc accatcataa agtccatctt 2460

gtaaaaaatg taccagataa tttagtatct gatacttact cagatggttc ttgcagcgga 2520

atcaaccgtt gtgatgaaca gcagcaggta gatatgcagc tagatgcgga gcatcatcca 2580

atggattgct gtgaagcggc tcaaacacat gagttttctt cctatattaa tacaggggat 2640

ctaaatgcaa gtgtagatca gggcatttgg gttgtattaa aagttcgaac aacagatggg 2700

tatgcgacgt taggaaatct tgaattggta gaggttgggc cattatcggg tgaatctcta 2760

gaacgcgaac aaagagataa tgcgaaatgg aatgcagagc taggaagaaa gcgtgcagaa 2820

acagatcgcg tgtatctagc tgcgaaacaa gcaattaatc atctatttgt agactatcaa 2880

gatcaacaat taaatccaga aattgggcta gcggaaataa atgaagcttc aaatcttgtg 2940

aagtcaattt cgggtgtata tagtgataca ctattacaga ttcctggaat taactacgaa 3000

atttacacag agttatccga tcgattacaa caagcatcgt atctgtatac gtctcgaaat 3060

gccgtgcaaa atggagactt taacagtggt ctagatagtt ggaatgcaac aacagatgca 3120

tcggttcagc aagatggcag tacacatttc ttagttcttt cgcattggga tgcacaagtt 3180

tcccaacaaa tgagagtaaa tttgaattgt aagtatgttt tacgtgtaac agcaaaaaaa 3240

gtaggaggcg gagatggata cgtcacaatc cgagatggcg ctcatcacca agaaactctt 3300

acatttaatg catgtgacta cgatgtaaat ggtacgtatg tcaatgacaa ttcgtacata 3360

acaaaagaag tggtattcta cccagagaca aaacatatgt gggtagaggt gagtgaatcc 3420

gaaggttcat tctatataga cagcattgag ttcattgaaa cacaagagta a 3471

<210> 3

<211> 1902

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaacaacg tcctcaacag cggcagggct acgaacggcg acgcgtacaa cgtggtcgcc 60

cacgacccct tctccttcca gcacaagagc ctcgacacca tccaggagga gtggatggag 120

tggaagaagg acaaccacat cctctacgtg gacccgatcg tgggcaccgt cgcctccttc 180

ctcctgaaga aggtcggcag cctcgtcgag aagcgcatcc tctccgagct gaggaacctc 240

atcttcccgt ccggcagcac gaacctcatg caggacatcc tgcgcgagac cgagaagttc 300

ctgaaccagc gcctcaacac ggacaccctg gctagggtca acgctgagct gaccggcctc 360

caggctaacg tcgaggagtt caaccgccag gtggacaact tcctcaaccc gaacaggaac 420

gccgtccccc tgtccatcac gtccagcgtg aacaccatgc agcagctctt cctgaacagg 480

ctcccccagt tccagatgca gggctaccag ctcctgctcc tgccactgtt cgctcaggct 540

gcgaacctcc acctgtcctt catccgcgac gtgatcctga acgctgacga gtggggcatc 600

agcgctgcta cgctcaggac ctaccagaac cacctgcgca actacacgag ggagtactcc 660

aactactgca tcaccacgta ccagacggcg ttccgcggcc tgaacaccag gctccacgac 720

atgctggagt tccgcaccta catgttcctc aacgtgttcg agtatgtgtc catctggagc 780

ctgttcaagt accagagcct cctggtctcc agcggcgcca acctctacgc ttccggcagc 840

ggcccacagc agacgcagtc cttcaccagc caggactggc cgttcctgta ctccctcttc 900

caggtgaaca gcaactacgt cctcaacggc ttctccggcg ctaggctgac gcagaccttc 960

ccaaacatcg tgggcctgcc aggcaccacg accacgcacg cgctcctggc tgctagggtg 1020

aactactccg gcggcgtctc cagcggcgac atcggcgctg tgcccttcaa ccagaacttc 1080

tcctgcagca cgttcctccc accactcctg accccattcg tccgcagctg gctggactcc 1140

ggcagcgaca ggggcggcat caacacggtg accaactggc agacggagag cttcgagacc 1200

acgctcggcc tgcgctccgg cgccttcacc gcgaggggca acagcaacta cttcccggac 1260

tacttcatcc gcaacatctc cggcgtgccc ctcgtggtca ggaacgagga cctccgcagg 1320

ccgctgcact acaaccagat caggaacatc gagtccccaa gcggcacccc aggcggcctc 1380

agggcgtaca tggtgagcgt ccataacagg aagaacaaca tctacgcggt ccacgagaac 1440

ggcacgatga tccacctggc cccggaggac tacacgggct tcaccatctc ccccatccac 1500

gcgacccagg tgaacaacca gacgcgcacc ttcatcagcg agaagttcgg caaccagggc 1560

gactccctca ggttcgagca gagcaacacc acggctaggt acaccctgag gggcaacggc 1620

aactcctaca acctctacct gcgcgtctcc agcatcggca acagcacgat ccgcgtgacc 1680

atcaacggca gggtctacac cgcctccaac gtgaacacca cgaccaacaa cgacggcgtg 1740

aacgacaacg gcgcgaggtt ctccgacatc aacatcggca acgtggtcgc cagcgacaac 1800

acgaacgtcc ccctcgacat caacgtgacg ctgaacagcg gcacccagtt cgagctgatg 1860

aacatcatgt tcgtgccgac caacctgccg cccctctact ga 1902

<210> 4

<211> 2205

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaaccgca acaaccagaa cgagtacgag atcatcgacg ctccacactg cggctgccca 60

agcgacgacg tggtcaagta cccactcgct tccgacccca acgctgctct gcagaacatg 120

aactacaagg actacctcca gacctacgac ggcgactaca cggacagcct catcaacccg 180

aacctgtcca tcaacaccag ggacgtgctg cagaccggca tcacgatcgt cggccgcatc 240

ctcggcttcc tgggcgtgcc gttcgctggc cagctcgtca ccttctacac gttcctcctg 300

aaccagctgt ggcccaccaa cgacaacgcc gtctgggagg cgttcatgga gcagatcgag 360

ggcatcatcg cccagaggat cagcgagcag gtggtccgca acgctctcga cgctctgacg 420

ggcatccacg actactacga ggagtacctc gccgcgctgg aggagtggct ggagaggcca 480

tccggcgcgc gggcaaacct ggcgttccag cgcttcgaga acctccacca gctgttcgtg 540

agccagatgc cgtccttcgg cagcggccca ggctccgaga gggacgccgt ggctctcctg 600

accgtctacg ctcaggctgc taacctccac ctcctgctcc tgaaggacgc cgagatctac 660

ggcgctcgct ggggcctcca gcagggccag atcaacctgt acttcaacgc ccagcaggac 720

cgcaccagga tctacacgaa ccactgcgtg gctacctaca acaggggcct gggcgacctg 780

aggggcacca acacggagag ctggctcaac taccaccagt tccgcaggga gatgacgctg 840

atggccatgg acctcgtcgc gctgttcccg tactacaacc tcaggcagta cccaaacggc 900

gctaacccac agctgacccg cgacgtgtac acggacccga tcgtcttcaa cccaagcgct 960

aacgtgggcc tctgcaggcg ctggggcaac aacccctaca acaccttctc cgagctggag 1020

aacgccttca tcaggccgcc ccacttcttc gaccgcctca actccctgac gatcagccgc 1080

aacaggttcg acgtgggcag caacttcatc gagccatggt ccggccacac cctcaggagg 1140

agcttcctga acacgtccgc tgtccaggag gacagctacg gccagatcac caaccagcgc 1200

accacgatca acctcccagc taacggcacc ggcagggtgg agagcacggc tgtcgacttc 1260

aggtccgctc tggtgggcat ctacggcgtc aacagggcca gcttcatccc aggcggcgtc 1320

ttcaacggca ccacgcagcc atccaccggc ggctgcaggg acctctacga ctccagcgac 1380

gagctgccac cagaggagtc cagcggcacc ttcgagcaca ggctcagcca cgtgacgttc 1440

ctgtccttca ccacgaacca ggctggctcc atcgctaacg ctggcagggt gccgacctac 1500

gtctggacgc acagggacgt cgacctcaac aacaccatca cggctgacag gatcacccac 1560

ctcccactga tcaagagcaa cgtgcagagg tccggcaggg ctgtcaaggg cccaggcttc 1620

acgggcggcg acgtgctgag gatgtccagc tccgacgccg acatcagcat catcggcatc 1680

accgctggcg ctccactcac gcagcagtac cgcatcaggc tgcgctacgc gtccaacgtg 1740

gacgtcacca tcaggctcgt gcgccaggac acgcagtcca acatcggcag catcaacctc 1800

ctgcgcacca tgaactccgg cgaggagagc cgctacgagt cctaccgcac cgtcgagatg 1860

cccggcaact tcaggatgac gagctccagc gcccagatca ggctcttcac ccagggcctg 1920

agggtgaacg gcgagctctt cctggacagc ctggagttca tcccagtcaa cccaacgagg 1980

gaggctgagg aggacctgga ggctgcgaag aaggcggtga ccagcctctt cacccgcacg 2040

tccgacggcc tgcagatcaa cgtgacggac taccaggtcg accaggccgc gaacctcgtg 2100

tcctgcctga gcgacgagca gtacggccac gacaagaaga tgctcctgga ggccgtcagg 2160

gctgctaagc gcctgtccag ggagcgcaac ctcctgcagg actga 2205

<210> 5

<211> 633

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Asn Asn Val Leu Asn Ser Gly Arg Ala Thr Asn Gly Asp Ala Tyr

1 5 10 15

Asn Val Val Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu Asp

20 25 30

Thr Ile Gln Glu Glu Trp Met Glu Trp Lys Lys Asp Asn His Ile Leu

35 40 45

Tyr Val Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys Lys

50 55 60

Val Gly Ser Leu Val Glu Lys Arg Ile Leu Ser Glu Leu Arg Asn Leu

65 70 75 80

Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu

85 90 95

Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg

100 105 110

Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe Asn

115 120 125

Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro Leu

130 135 140

Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg

145 150 155 160

Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu

165 170 175

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

180 185 190

Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr

195 200 205

Gln Asn His Leu Arg Asn Tyr Thr Arg Glu Tyr Ser Asn Tyr Cys Ile

210 215 220

Thr Thr Tyr Gln Thr Ala Phe Arg Gly Leu Asn Thr Arg Leu His Asp

225 230 235 240

Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val

245 250 255

Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly

260 265 270

Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe

275 280 285

Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser

290 295 300

Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Thr Gln Thr Phe

305 310 315 320

Pro Asn Ile Val Gly Leu Pro Gly Thr Thr Thr Thr His Ala Leu Leu

325 330 335

Ala Ala Arg Val Asn Tyr Ser Gly Gly Val Ser Ser Gly Asp Ile Gly

340 345 350

Ala Val Pro Phe Asn Gln Asn Phe Ser Cys Ser Thr Phe Leu Pro Pro

355 360 365

Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg

370 375 380

Gly Gly Ile Asn Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Thr

385 390 395 400

Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn

405 410 415

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

420 425 430

Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Gln Ile Arg

435 440 445

Asn Ile Glu Ser Pro Ser Gly Thr Pro Gly Gly Leu Arg Ala Tyr Met

450 455 460

Val Ser Val His Asn Arg Lys Asn Asn Ile Tyr Ala Val His Glu Asn

465 470 475 480

Gly Thr Met Ile His Leu Ala Pro Glu Asp Tyr Thr Gly Phe Thr Ile

485 490 495

Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile

500 505 510

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

515 520 525

Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn

530 535 540

Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr

545 550 555 560

Ile Asn Gly Arg Val Tyr Thr Ala Ser Asn Val Asn Thr Thr Thr Asn

565 570 575

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

580 585 590

Gly Asn Val Val Ala Ser Asp Asn Thr Asn Val Pro Leu Asp Ile Asn

595 600 605

Val Thr Leu Asn Ser Gly Thr Gln Phe Glu Leu Met Asn Ile Met Phe

610 615 620

Val Pro Thr Asn Leu Pro Pro Leu Tyr

625 630

<210> 6

<211> 734

<212> PRT

<213> Bacillus thuringiensis (Bacillus thuringiensis)

<400> 6

Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His

1 5 10 15

Cys Gly Cys Pro Ser Asp Asp Val Val Lys Tyr Pro Leu Ala Ser Asp

20 25 30

Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Thr

35 40 45

Tyr Asp Gly Asp Tyr Thr Asp Ser Leu Ile Asn Pro Asn Leu Ser Ile

50 55 60

Asn Thr Arg Asp Val Leu Gln Thr Gly Ile Thr Ile Val Gly Arg Ile

65 70 75 80

Leu Gly Phe Leu Gly Val Pro Phe Ala Gly Gln Leu Val Thr Phe Tyr

85 90 95

Thr Phe Leu Leu Asn Gln Leu Trp Pro Thr Asn Asp Asn Ala Val Trp

100 105 110

Glu Ala Phe Met Glu Gln Ile Glu Gly Ile Ile Ala Gln Arg Ile Ser

115 120 125

Glu Gln Val Val Arg Asn Ala Leu Asp Ala Leu Thr Gly Ile His Asp

130 135 140

Tyr Tyr Glu Glu Tyr Leu Ala Ala Leu Glu Glu Trp Leu Glu Arg Pro

145 150 155 160

Ser Gly Ala Arg Ala Asn Leu Ala Phe Gln Arg Phe Glu Asn Leu His

165 170 175

Gln Leu Phe Val Ser Gln Met Pro Ser Phe Gly Ser Gly Pro Gly Ser

180 185 190

Glu Arg Asp Ala Val Ala Leu Leu Thr Val Tyr Ala Gln Ala Ala Asn

195 200 205

Leu His Leu Leu Leu Leu Lys Asp Ala Glu Ile Tyr Gly Ala Arg Trp

210 215 220

Gly Leu Gln Gln Gly Gln Ile Asn Leu Tyr Phe Asn Ala Gln Gln Asp

225 230 235 240

Arg Thr Arg Ile Tyr Thr Asn His Cys Val Ala Thr Tyr Asn Arg Gly

245 250 255

Leu Gly Asp Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Asn Tyr His

260 265 270

Gln Phe Arg Arg Glu Met Thr Leu Met Ala Met Asp Leu Val Ala Leu

275 280 285

Phe Pro Tyr Tyr Asn Leu Arg Gln Tyr Pro Asn Gly Ala Asn Pro Gln

290 295 300

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

305 310 315 320

Asn Val Gly Leu Cys Arg Arg Trp Gly Asn Asn Pro Tyr Asn Thr Phe

325 330 335

Ser Glu Leu Glu Asn Ala Phe Ile Arg Pro Pro His Phe Phe Asp Arg

340 345 350

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

355 360 365

Phe Ile Glu Pro Trp Ser Gly His Thr Leu Arg Arg Ser Phe Leu Asn

370 375 380

Thr Ser Ala Val Gln Glu Asp Ser Tyr Gly Gln Ile Thr Asn Gln Arg

385 390 395 400

Thr Thr Ile Asn Leu Pro Ala Asn Gly Thr Gly Arg Val Glu Ser Thr

405 410 415

Ala Val Asp Phe Arg Ser Ala Leu Val Gly Ile Tyr Gly Val Asn Arg

420 425 430

Ala Ser Phe Ile Pro Gly Gly Val Phe Asn Gly Thr Thr Gln Pro Ser

435 440 445

Thr Gly Gly Cys Arg Asp Leu Tyr Asp Ser Ser Asp Glu Leu Pro Pro

450 455 460

Glu Glu Ser Ser Gly Thr Phe Glu His Arg Leu Ser His Val Thr Phe

465 470 475 480

Leu Ser Phe Thr Thr Asn Gln Ala Gly Ser Ile Ala Asn Ala Gly Arg

485 490 495

Val Pro Thr Tyr Val Trp Thr His Arg Asp Val Asp Leu Asn Asn Thr

500 505 510

Ile Thr Ala Asp Arg Ile Thr His Leu Pro Leu Ile Lys Ser Asn Val

515 520 525

Gln Arg Ser Gly Arg Ala Val Lys Gly Pro Gly Phe Thr Gly Gly Asp

530 535 540

Val Leu Arg Met Ser Ser Ser Asp Ala Asp Ile Ser Ile Ile Gly Ile

545 550 555 560

Thr Ala Gly Ala Pro Leu Thr Gln Gln Tyr Arg Ile Arg Leu Arg Tyr

565 570 575

Ala Ser Asn Val Asp Val Thr Ile Arg Leu Val Arg Gln Asp Thr Gln

580 585 590

Ser Asn Ile Gly Ser Ile Asn Leu Leu Arg Thr Met Asn Ser Gly Glu

595 600 605

Glu Ser Arg Tyr Glu Ser Tyr Arg Thr Val Glu Met Pro Gly Asn Phe

610 615 620

Arg Met Thr Ser Ser Ser Ala Gln Ile Arg Leu Phe Thr Gln Gly Leu

625 630 635 640

Arg Val Asn Gly Glu Leu Phe Leu Asp Ser Leu Glu Phe Ile Pro Val

645 650 655

Asn Pro Thr Arg Glu Ala Glu Glu Asp Leu Glu Ala Ala Lys Lys Ala

660 665 670

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

675 680 685

Thr Asp Tyr Gln Val Asp Gln Ala Ala Asn Leu Val Ser Cys Leu Ser

690 695 700

Asp Glu Gln Tyr Gly His Asp Lys Lys Met Leu Leu Glu Ala Val Arg

705 710 715 720

Ala Ala Lys Arg Leu Ser Arg Glu Arg Asn Leu Leu Gln Asp

725 730

Claims (10)

1. The gene cry9Ee is artificially synthesized, and the nucleotide sequence is shown in SEQ ID NO. 4.

2. A plant expression vector comprising cry9Ee of claim 1.

3. The expression vector of claim 2, named plant expression vector pC9EG, and its skeleton vector is pCAMBIA2300, and its structure is shown in FIG. 2.

4. Use of the expression vector of claim 2 or 3 in a pest-resistant plant.

The application of the Cry2Ah-vp and Cry9Ee protein combination in insect-resistant plants, wherein the amino acid sequence of the Cry2Ah-vp protein is shown as SEQ ID NO.5, and the amino acid sequence of the Cry9Ee protein is shown as SEQ ID NO. 6.

6. The use according to claim 5, for transforming plants with expression vectors containing Cry2Ah-vp gene and Cry9Ee gene to express Cry2Ah-vp and Cry9Ee proteins, thereby making the transformed plants insect-resistant.

7. The use of claim 5, wherein the Cry2Ah-vp and Cry9Ee proteins are expressed by different plants by transforming the expression vectors respectively containing Cry2Ah-vp gene and Cry9Ee gene into the same plant, and then the Cry2Ah-vp and Cry9Ee genes are polymerized into the same plant by crossing method, so that the hybrid plant expresses Cry2Ah-vp and Cry9Ee proteins, thereby making the hybrid plant have insect-resistant property.

8. The use of claim 7, wherein the expression vector containing cry2Ah-vp gene has the nucleotide sequence of cry2Ah-vp gene shown in SEQ ID NO.3, and the expression vector containing cry9Ee gene is the vector of claim 2 or 3.

9. The use according to claim 8, wherein the expression vector plant containing cry2Ah-vp gene is pC2HBvp and the backbone vector is pCAMBIA 3300.

10. The use according to any one of claims 4 to 9, wherein the pest resistance is against a lepidopteran pest.

Images