CN114032247B - Application of insecticidal gene cry2Ah-vp and cry9Ee combination in insect-resistant plants - Google Patents

Abstract

The invention relates to an application of insecticidal gene cry2Ah-vp and cry9Ee combination in insect-resistant plants. The invention modifies cry2Ah-vp and cry9Ee genes with high toxicity to lepidoptera pests and synthesizes a new DNA sequence, and uses an agrobacterium-mediated method to transfer the two genes into corn respectively, and screens to obtain 2HVB5 and 9EG1 transformation events with high resistance to myxoma and corn borer. And combining cry2Ah-vp and cry9Ee genes by a gene polymerization method to obtain the 2HVB5/9EG1 transgenic corn material. Experimental results show that the transgenic corn combined by cry2Ah-vp and cry9Ee genes has high insecticidal activity on armyworms and corn borers, especially resistant corn borers. The invention has very important significance for cultivating insect-resistant plants, pest control and resistance control by utilizing the cooperative expression of cry2Ah-vp and cry9Ee genes to transform plants.

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

Background

Insect pests are an important factor in the reduction of crop yield in the world, and on average, about 10% of the total grain yield is lost annually, with direct economic losses of up to several billion dollars. The control of the past decades mainly depends on chemical pesticides, and the chemical pesticides have serious consequences such as environmental pollution, human and animal poisoning, ecological imbalance and the like while making great contribution to agricultural production. The development and application of the transgenic technology provide a safe and effective new method for crop insect resistance.

Bacillus thuringiensis (Bacillus thuringiensis, bt for short) is an extremely widespread gram-positive bacterium. It produces protein-like companion crystals (parasporal crystal) simultaneously with the formation of spores, and has insecticidal activity specific for a wide variety of insects, as well as nematodes, mites and protozoa (Schnepf E.et al 1998,Microbiology and Molecular Biology Review,62 (3): 775-806). The insecticidal crystal protein (Insecticidal Crystal Proteins, ICPs) is also called Delta-endotoxin, is first dissolved in the midgut of insects to become protoxin, then degraded into toxin with specific activity by intestinal protease, and combined with specific receptors on the midgut (Craig, 2007,Microbiology and Molecular Biology Review,71 (2): 255-281), so that insects die. The bacillus thuringiensis insecticidal crystal protein has good insecticidal effect, is harmless to human and livestock and does not pollute the environment, so that the bacillus thuringiensis insecticidal crystal protein is widely applied to biological control of pests.

Since commercialization of transgenic crops in 1996, the application of transgenic crops has grown rapidly, and 29 countries worldwide planted 1.904 hundred million hectares of transgenic crops in 2019, the planting area of transgenic crops has increased by about 112 times than 1996 (isana Briefs, 2019). However, the types of the insect-resistant genes of the transgenic insect-resistant crops commercialized at present are relatively single, and mainly cry 1A. The long-time large-area popularization and planting have the risks of reducing pest refuge and increasing pest resistance. There is therefore a need to use new highly virulent insecticidal genes or new combinations of genes to avoid the risk of increased resistance to pests.

The Cry2Ah gene is obtained by mixing and collecting soil samples without culturing, and the Cry2Ah protein has weight inhibition activity on corn borers, growth inhibition activity on cotton bollworms, and good growth inhibition activity on Cry1 Ac-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). Inserting a proline behind valine at position 354 of Cry2Ah to obtain mutant Cry2Ah-vp. After the Cry2Ah and Cry2Ah-vp genes are respectively transferred into tobacco plants, experiments of the insecticidal activity of cotton bollworms and Cry1Ac resistant cotton bollworms are carried out, and the results show that Cry2Ah-vp has higher insecticidal activity compared with 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 has described in detail the cry2Ah-vp gene sequence, vector and application thereof in patent 'artificially synthesized insecticidal gene with high toxicity to lepidoptera pests' (patent number: ZL 201710145166.6) and patent 'cry 2Ah-vp gene application in resisting myxose' (patent number: ZL201910191397. X).

The Cry9Ee gene is cloned from bacillus thuringiensis T03B001 strain, cry9Ee has no resistance to Cry1A, and has high toxicity to plutella xylostella, corn borer and Cry1Ac resistant plutella xylostella (Shu C L, su H Q, zhang J, he K L, huang D F, song F P.2013, appl Microbiol Biotechnol, 97:9705-9713).

The monovalent Bt insecticidal genes can face the problems of limited toxicity, narrow insecticidal spectrum and resistance to pests in application, and researchers can achieve the effects of expanding the insecticidal spectrum and controlling the resistance by using the combined application of two or more Bt insecticidal genes. However, different combinations of Bt genes produce different synergistic and antagonistic results due to different acting receptors, and the Zhang Jie research group found that Bt proteins Cry1Ai and Cry9Aa, cry1Ea and Cry9Aa have a synergistic effect, whereas the Cry1Ai and Cry1Ea combinations have no obvious synergistic effect (Cai Jilin, buddha, song Fuping, huang Bo, zhang Jie.2013, plant protection, 39 (1): 66-70); studies by 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 both Cry1 and Cry2 did not find a synergistic effect (Yang J, quan Y, sivaprapass P, shabbir M, wang Z, ferre J, he k.2018, toxins, 10:454). The selection of gene combinations with synergistic effects is a new source of genes for creating new generation insect-resistant plants.

Disclosure of Invention

Aiming at the requirements in the field, the invention provides the application of two Cry2Ah-vp and Cry9Ee gene combinations to the control of lepidoptera pests, and the Cry2Ah-vp and Cry9Ee genes are subjected to codon optimization and synthesized into new DNA sequences to construct plant expression vectors to transform corn, and the Cry2Ah-vp and Cry9Ee genes are combined together by a gene polymerization method, so that experiments show that the synergistic expression of the Cry2Ah-vp and Cry9Ee genes endows corn with the effect of giving high-resistance to the corn borers, corn borers and Cry1A type resistance corn borers, and the application prospect in pest resistance management is provided.

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

The plant expression vector is pC2HBvp, and the skeleton vector is pCAMBIA3300.

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

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

The plant expression vector is pC9EG, and the skeleton vector is pCAMBIA2300.

The structure of the plant expression vector pC9EG is shown in FIG. 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 an expression vector containing Cry2Ah-vp genes and Cry9Ee genes into plants so as to express Cry2Ah-vp and Cry9Ee proteins, thereby enabling the transformed plants to have insect-resistant characteristics.

In order to transform different plants respectively containing Cry2Ah-vp genes and Cry9Ee genes, the Cry2Ah-vp and Cry9Ee proteins are expressed by the different plants respectively, then Cry2Ah-vp and Cry9Ee genes are polymerized into the same plant by a hybridization method, so that the Cry2Ah-vp and Cry9Ee proteins are expressed by the hybrid plants, and the hybrid plants have insect-resistant characteristics.

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

The expression vector plant containing cry2Ah-vp gene is pC2HBvp, and the skeleton vector is pCAMBIA3300.

The insect resistance is lepidopteran pest resistance.

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

The invention discloses a method for preparing a plant expression vector pC2HBvp and pC9EG, which comprises the steps of carrying out codon optimization and mutation on a nucleotide sequence of cry2Ah (SEQ ID NO. 1), carrying out codon optimization on the nucleotide sequence of cry9Ee (SEQ ID NO. 2), synthesizing a novel gene cry2Ah-vp (SEQ ID NO. 3) and cry9Ee (SEQ ID NO. 4) in a synthetic mode, respectively constructing plant expression vectors pC2HBvp and pC9EG containing cry2Ah-vp and cry9Ee, transferring the plant expression vectors into corn by using an agrobacterium-mediated method, and carrying out PCR identification and insect resistance experiments to obtain a cry2Ah-vp gene corn event 2HVB5 and a cry9Ee gene corn event 9EG1. The two transgenic events are polymerized into the same corn plant by a hybridization method, and PCR detection results show that the cry2Ah-vp and cry9Ee genes are integrated into the corn genome. The insect resistance experiment result shows that the transgenic corn combined by Cry2Ah-vp and Cry9Ee genes has good insecticidal activity on the armyworms and the corn borers, especially on Cry1Ab resistant corn borers and Cry1Ac resistant corn borers, and the armyworms, the corn borers, the Cry1Ab resistant corn borers and the Cry1Ac resistant corn borers die after 3 days of insect inoculation.

The combined application of cry2Ah-vp and cry9Ee genes has good insecticidal activity on armyworms, corn borers and resistant corn borers, which is helpful for reducing the risk of rising of pest resistance, and the transgenic corn with combined application is a germplasm resource with good corn pest-resistant breeding and pest control, and has good commercial prospect in corn pest-resistant breeding.

Drawings

FIG. 1 schematic construction of plant expression vector pC2 HBvp.

FIG. 2 construction of plant expression vector pC9 EG.

FIG. 3 PCR detection of cry2Ah-vp gene of corn transformed with pC2HBvp vector

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

FIG. 4 PCR detection of the pC9EG vector maize cry9Ee Gene

Wherein M is DNA molecular weight standard Super Marker; CK+: amplifying the product by taking plasmid pC9EG as a template; CK-: is the product of amplification by taking non-transgenic corn genome DNA as a template; 0: blank, using water as template amplification product; 1-5: part of cry9Ee gene-transferred 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 cry2Ah-vp gene (A), bar gene (B), cry9Ee gene (C) and GAT gene (D);

wherein M is DNA molecular weight standard Super Marker; CK+: amplifying the product by taking plasmid pC2HBvp or pC9EG as a template; CK-: is the product of amplification by taking non-transgenic corn genome DNA as a template; 0: blank, using water as template amplification product; 1-3: transgenic corn genome DNA combined by 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 biological materials referred to below are all stored in the laboratory of the applicant and can be dispensed externally.

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

Bt cry2Ah (GenBank No. ACL 13555.1) is a gene cloned from Bt strains isolated in China, a coding region of the gene has 1899bp, a nucleic acid sequence is shown in SEQ ID NO.1, and a coded protein consists of 632 amino acids. The Cry2Ah-vp mutant protein is obtained by adding a proline (Pro) to a valine (Val) at position 354 of a Cry2Ah1 protein, adding 3 bases CCC after 1062bp of a nucleotide sequence, encoding the proline, and optimizing a Cry2Ah-vp nucleotide sequence shown in SEQ ID NO.3 and an amino acid sequence 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 encoded protein consists of 1156 amino acids. By analysis of the Cry9Ee protein sequence domain, there are three conserved domains: amino acids 86-293 are domain I (endotoxin_N), amino acids 302-508 are domain II (endotoxin_M) and amino acids 510-657 are domain III (endotoxin_C) are essential segments of activity. In the present invention, the coding sequence (2202 bp) of amino acids 1-734 of Bt cry9Ee gene was selected and optimized according to plant preferred codons. The GC content of the original cry9Ee gene (2202 bp) is 39.2%, the GC content of the modified gene is 63.5%, the similarity with the original cry9Ee nucleic acid sequence is 66.53%, the optimized cry9Ee nucleic acid sequence is shown as SEQ ID NO.4, and the amino acid sequence is shown as 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 a NOS terminator, the construction process is described in detail in patent 'cry 2Ah-vp gene application in insect resistance' (patent number: ZL201910191397. X), a 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 synthesized artificially, 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 (plasmid was stored in the national institute of agricultural science and biotechnology Lang Zhihong subject group, which was available to the public), and the synthesized OMK-cry9Ee fragment was ligated into pUC57-UN vector by BamHI and KpnI cleavage, and cry9Ee expression cassette was ligated into pCGAT vector by HindIII and EcoRI cleavage. The pCGAT intermediate vector is a vector obtained by adding an OMK sequence for enhancing gene expression to the upstream of the artificially synthesized and optimized glyphosate acetylase GAT gene, cutting the nptII gene in T-DNA by XhoI digestion of a commercial vector pCAMBIA2300, and inserting a synthesized OMK-GAT fragment into the pCAMBIA2300 in a seamless cloning mode (the plasmid is stored in a institute of biotechnology Lang Zhihong of China academy of agricultural sciences and can be provided for the public). The expression cassette containing cry9Ee gene is connected with pCGAT to obtain final vector pC9EG, the schematic diagram of the vector is shown in figure 2, and the vector contains glyphosate acetyl transferase gene GAT and insecticidal gene cry9Ee.

3. Acquisition of cry2Ah-vp and cry9Ee transgenic maize

The vectors pC2HBvp and pC9EG are respectively transformed into agrobacterium EHA105 by a freeze thawing method, and PCR is carried out for identification. Taking freshly stripped maize immature embryo with the thickness of about 1.2mm as a material, placing the maize immature embryo into an infection culture medium for one hour, washing once with the infection culture medium, immersing the maize immature embryo into an agrobacterium liquid added with 100 mu M acetosyringone, and placing for 5 minutes. Taking out, sucking with sterilized filter paper, placing on co-culture medium, and making into dark strip at 26deg.CCo-culture was performed under the pieces for 3 days, and control was set. Transferring young embryos to a recovery culture medium to culture for 10 days until callus is induced, then removing buds of the callus, transferring the callus to a screening culture medium containing a corresponding screening agent, carrying out subculture every two weeks, transferring the resistant callus to a regeneration culture medium to differentiate by visible light after 6 weeks, starting to appear green buds after about one week of visible light, cutting the callus to separate the green buds and transferring the green buds to the regeneration culture medium to culture, facilitating the growth of main stems, transferring the main stems to the regeneration culture medium to induce rooting when the main stems are elongated to 3-4 cm, and transferring the maize plants to a greenhouse small flowerpot to grow after the maize plants grow to be thick and have developed root systems. After two weeks of continuous culture, transferring the transformed seedlings to a greenhouse after the transformed seedlings grow well, sleeving the transformed seedlings by a paper bag after the female ears are threaded, pollinating the transformed seedlings after the male ears are loose in powder, and harvesting fruits. By corn transformation, 31 strains of the pC2 HBvp-transferred carrier are obtained, and 91 strains of the pC9 EG-transferred carrier are obtained. The transformed plants were verified by PCR, and the primer sequences are shown in Table 1. mu.L of the reaction mixture was used to contain 100ng of DNA template, 0.1. Mu.M of each primer, 10. Mu.L of 2×taq Master mix (Dye), ddH ₂ O was made up to 20. Mu.L. The cry2Ah-vp reaction conditions are: 94℃for 5min, (94℃for 30s,56℃for 30s,72℃for 60 s) 30 cycles, 72℃for 5min. The cry9Ee reaction conditions were: 94℃for 5min, (94℃for 30s,56.9℃for 30s,72℃for 30 s) 30 cycles, 72℃for 5min. The PCR detection result of the cry2Ah-vp transgenic corn is shown in FIG. 3, and the PCR detection result of the cry9Ee transgenic corn is shown in FIG. 4. The results show that the exogenous gene cry2Ah-vp or cry9Ee gene has been introduced into the maize genome.

The culture medium:

infection culture solution: n6 salts and N6 vitamins (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, stored at 4 ℃; before use, filter sterilized Acetosyringone (AS) was added to a final concentration of 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 vegetable gel (pH 5.8), and after autoclaving, silver nitrate with a final concentration of 0.85mg/L, 100. Mu.M AS,300mg/L cysteine were added;

recovery 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 vegetable gel (pH 5.8), and after autoclaving, silver nitrate with a final concentration of 0.85mg/L and 200mg/L carbenicillin were added after filter sterilization;

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

regeneration medium: MS salt and MS vitamin, 30g/L sucrose, 100mg/L inositol, 3g/L plant gel (pH 5.8), and autoclaving.

The maize young embryo is a freshly peeled 1.2mm long young embryo.

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 corn

After sowing the obtained cry2Ah-vp and cry9Ee transgenic corn materials, carrying out indoor bioassays when corn grows to a six-leaf period. And (5) determining the insecticidal activity of the transgenic corn plants on the armyworms and the sensitive corn borer larvae. Indoor bioassay of myxoplasma and sensitive corn borer larvae using 24-well Petri dishes, 1cm was taken ² Corn leaves of the size are placed in 24 holes, one head of the 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 inbred Zheng 58 material as controls. Screening to obtain cry2Ah-vp transgenic corn material 2HVB5 and cry9Ee transgenic corn material 9EG1 with good insect resistance. The insect test result shows that the mortality rate of the 2HVB5 material is 100% after 4 days of insect inoculation, and the mortality rate of the 2HVB5 material is 100% after 5 days of insect inoculation; after 4 days of insect inoculation, all the corn borers of the 9EG1 material die; the insecticidal activity of 9EG1 material on the mythic fungus is lower than that of 2HVB5 material, the corrected mortality rate of the mythic fungus after 5 days of insect inoculation is 53.56%, and the mythic fungus is tested individually after 6 days of insect inoculationInsect survival (table 2). Indoor biological activity identification shows that the 2HVB5 has good insecticidal activity on armyworms and corn borers, and the 9EG1 material has good insecticidal activity on corn borers.

TABLE 2 correction mortality statistics for corn borers and armyworms by 2HVB5 and 9EG1

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

Bt cry2Ah-vp gene and cry9Ee gene belong to cry2 type and cry9 type genes, and the amino acid sequence similarity is 9.07%. The 2HVB5/9EG1 combined material containing two genes of cry2Ah-vp and cry9Ee is obtained by combining the transgenic corn 2HVB5 and 9EG1 through a hybridization method. PCR was performed on 2HVB5/9EG1 material using cry2Ah-vp, bar, cry Ee and GAT gene specific primers, respectively, the primer sequences are shown in Table 1. mu.L of the reaction mixture was used to contain 100ng of DNA template, 0.1. Mu.M of each primer, 10. Mu.L of 2×taq Master mix (Dye), ddH ₂ O was made up to 20. Mu.L. The cry2Ah-vp reaction conditions are: 94℃for 5min, (94℃for 30s,56℃for 30s,72℃for 60 s) 30 cycles, 72℃for 5min. The bar reaction conditions were: 94℃for 5min, (94℃for 30s,56℃for 30s,72℃for 20 s) 30 cycles, 72℃for 5min. The cry9Ee reaction conditions were: 94℃for 5min, (94℃for 30s,56.9℃for 30s,72℃for 30 s) 30 cycles, 72℃for 5min. The GAT reaction conditions were: 94℃for 5min, (94℃for 30s,58.5℃for 30s,72℃for 20 s) for 30 cycles, 72℃for 5min. The PCR detection results are shown in FIG. 5. The results showed that cry2Ah-vp, bar, cry Ee and GAT genes could be detected in the 2HVB5/9EG1 material, indicating that the foreign gene was integrated into the same material.

The PCR detection of 2HVB5/9EG1 material containing 4 genes is subjected to indoor bioassay, and the insecticidal activity of 2HVB5/9EG1 material combined by Cry2Ah-vp and Cry9Ee genes on larvae of armyworms, corn borers, cry1Ab resistant corn borers and Cry1Ac resistant corn borers is detected. The insect test results are shown in Table 3, and after 3 days of insect inoculation, the corrected mortality rates of the armyworms, the corn borers, the Cry1Ab resistant corn borers and the Cry1Ac resistant corn borers of the 2HVB5/9EG1 material are all 100%, which shows that the Cry2Ah-vp and Cry9Ee gene combination has very high insecticidal activity on the armyworms, the corn borers, the Cry1Ab resistant corn borers and the Cry1Ac resistant corn borers. Although 2HVB5 containing cry2Ah-vp gene alone and 9EG1 containing cry9Ee gene alone had good insecticidal activity against corn borers, individual insects survived 3 days after inoculation and all tested insects died after 4 or 5 days. The statistical analysis results show that the three materials of 2HVB5/9EG1, 2HVB5 and 9EG1 have extremely significant differences on the corrected mortality of corn borers (2 HVB5/9EG1>2HVB 5). The 2HVB5 material has good insecticidal activity on the mythic fungus, the corrected death rate of the mythic fungus after 3 days of insect inoculation is 94.38%, the insecticidal activity of the 9EG1 material on the mythic fungus is general, the corrected death rate of the mythic fungus after 3 days of insect inoculation is 39.49%, and the statistical analysis result shows that the corrected death rates of the three materials of 2HVB5/9EG1, 2HVB5 and 9EG1 have extremely obvious difference (2 HVB5/9EG1>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 Cry1Ab resistant corn borers is obviously different from that of the 9EG1 material, is obviously different from that of the 2HVB5 material, and is not obviously different from that of the 2HVB5 material; the corrected mortality rate of 2HVB5/9EG1 for Cry1Ac resistant corn borers is not significantly different from that of 9EG1 material, is significantly different from that of 2HVB5 material, and is significantly different from that of 9EG1 and 2HVB5 materials.

TABLE 3 correction mortality statistics for armyworms, corn borers and resistant corn borers with 2HVB5/9EG1 materials

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

Experimental results show that the cry2Ah-vp and cry9Ee gene combination application has high insecticidal activity on armyworms and corn borers, especially resistant corn borers, and is superior to a single gene. The cry2Ah-vp and cry9Ee gene combination can be used for breeding of insect-resistant corn, has very important significance for corn pest control and resistance management, and has good industrialization prospect.

Sequence listing

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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 (6)

1. The application of the combination of Cry2Ah-vp and Cry9Ee in preparing insect-resistant transgenic plants, wherein the amino acid sequence of the Cry2Ah-vp protein is shown as SEQ ID NO.5, the amino acid sequence of the Cry9Ee protein is shown as SEQ ID NO.6, and the insect is insect-resistant and corn borer-resistant.
2. 2. The use according to claim 1, for the transformation of plants with expression vectors containing Cry2Ah-vp gene and Cry9Ee gene, such that they express Cry2Ah-vp and Cry9Ee proteins, whereby the transformed plants have insect-resistant properties.
3. 3. The use according to claim 1, wherein the Cry2Ah-vp and Cry9Ee genes are polymerized into the same plant by crossing to make the hybrid plant express the Cry2Ah-vp and Cry9Ee proteins, so that the hybrid plant has insect-resistant property.
4. 4. The use according to claim 3, wherein the nucleotide sequence of the cry2Ah-vp gene in the cry2Ah-vp gene-containing expression vector is shown in SEQ ID NO.3, and the nucleotide sequence of the cry9Ee gene in the cry9Ee gene-containing expression vector is shown in SEQ ID NO. 4.
5. 5. The use according to claim 4, wherein the expression vector containing cry2Ah-vp gene is pC2HBvp and the skeleton vector is pCAMBIA3300.
6. 6. The use according to claim 4, wherein the expression vector containing cry9Ee gene is pC9EG and the backbone vector is pCAMBIA2300.

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