Insect-resistant protein mutant Cry2AhM, fused insect-resistant protein FP2Ah1Ac, expression vector and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an insect-resistant protein mutant Cry2AhM, a fused insect-resistant protein FP2Ah1Ac, an expression vector and application. The amino acid sequence of the insect-resistant protein mutant Cry2AhM provided by the invention is as shown in SEQ ID No.1, and the insect-resistant protein mutant Cry2AhM has high insecticidal activity on lepidoptera pests; according to the fusion insect-resistant protein FP2Ah1Ac provided by the invention, the mutant and GFM Cry1A protein are subjected to fusion expression, and the resistance of lepidoptera pests to transgenic products of the lepidoptera pests can be reduced, so that the lepidoptera pests are efficiently killed, and normal growth of plants is not influenced; according to the expression vector provided by the invention, the fusion insect-resistant protein FP2Ah1Ac can be highly expressed in a plant body, so that receptor cotton obtains the character of high cotton bollworm resistance, and the fusion insect-resistant protein FP2Ah1Ac has important significance in reducing insect damage, reducing pesticide use, reducing production cost and improving cotton yield.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology, and particularly relates to a mutant Cry2AhM of insect-resistant protein, a fusion insect-resistant protein FP2Ah1Ac, an expression vector and application. BACKGROUND

[0002] Bt protein (Bacillus thuringiensis insecticidal crystal protein) is a kind of efficient, safe and widely used biopesticide protein, which can specifically kill Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera and Mallophaga, etc. Agricultural pests, and is harmless to non-target insects, birds and humans and animals. Bt protein can combine with specific receptors in the epithelial cells of the target insect gut, leading to cell membrane perforation, and ultimately causing insect death. By introducing Bt gene into plants, the plants can be endowed with specific insect resistance, thus effectively preventing and controlling major agricultural pests, and having important economic value and application prospect. In the 1990s, the Monsanto company in the United States introduced Cry1Ac gene and Cry1Ab gene into cotton and corn, and cultivated transgenic insect-resistant cotton and corn for commercial application, which not only reduced the dependence on chemical pesticides in agricultural production, but also significantly improved the yield and economic benefits of crops. In 1992, researcher Guo Sandui of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, introduced the artificially synthesized GFM Cry1A gene into cotton, and cultivated the first generation of domestic insect-resistant cotton, which was gradually industrialized and applied. As of 2020, the planting area of transgenic insect-resistant cotton in China accounted for more than 98% of the total area, which greatly reduced the threat of cotton bollworm and other pests to cotton production.

[0003] So far, there have been cry1Ac , GFM cry1Ab / c , cry2Ab2 , cry1F , vip3Aa , cry1Ab , cry2Ae and mcry51Aa2A total of 8 insect-resistant genes have been introduced into cotton and have been commercialized (https: / / www.isaaa.org / gmapprovaldatabase / ). However, with the large-scale promotion of transgenic insect-resistant crops, the problem of target pests resistant to Bt proteins has become increasingly prominent. For example, Tabashnik et al. (Nature Biotechnology, 2008) found that lepidopteran pests such as cotton bollworms are extremely susceptible to developing resistance to single Bt protein-containing transgenic cotton after long-term feeding, which further proves that the widespread use of single insect-resistant genes increases the risk of pest resistance. According to the report of the International Service for the Acquisition of Agri-Biotech Applications (ISAAA), from 2010 to 2020, a number of countries around the world have reported cases of pests resistant to single Bt proteins, and the resistance problem has become a major obstacle to the sustainable application of transgenic insect-resistant crops.

[0004] In view of the above problem of insect-resistant cotton production, Liao et al. (Journal of Invertebrate Pathology, 2002) found through the functional study of the Bt insecticidal protein family that different Bt proteins have different insecticidal mechanisms against the same pest, and the pest is not susceptible to cross-resistance to two different Bt proteins. Therefore, by co-expressing two or more Bt proteins, it is possible to not only improve the insecticidal activity of transgenic insect-resistant crops, but also delay the development of pest resistance, thereby providing technical support for the sustainable application of insect-resistant transgenic crops. However, multi-gene co-expression may not achieve ideal insecticidal activity due to low expression efficiency, may cause abnormal plant growth and development due to protein toxicity, and may reduce the overall insecticidal activity due to protein antagonism. SUMMARY

[0005] In view of the above problem, the present application provides an insect-resistant protein mutant Cry2AhM, a fusion insect-resistant protein FP2Ah1Ac, an expression vector and an application. The insect-resistant protein mutant Cry2AhM provided by the present application has high insecticidal activity against lepidopteran pests and can be used to prepare a fusion insect-resistant protein with high insecticidal activity; the fusion insect-resistant protein FP2Ah1Ac provided by the present application fuses and expresses the mutant and GFM Cry1A protein, which can reduce the resistance of lepidopteran pests to its transgenic product, thereby efficiently killing the pests and not affecting the normal growth of the plant; the expression vector provided by the present application enables the fusion insect-resistant protein FP2Ah1Ac to be highly expressed in the plant body, thereby obtaining a transgenic plant with excellent insecticidal activity.

[0006] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions: The application provides a mutant Cry2AhM insect-resistant protein, and the amino acid sequence of the mutant Cry2AhM insect-resistant protein is shown as SEQ ID No. 1.

[0007] The Cry2Ah protein (GenBank: KX034204.1) has an unsatisfactory insect-resistant activity on pests such as cotton bollworms that threaten cotton production at the present stage, and the mutant Cry2AhM insect-resistant protein is obtained by mutating the amino acid sequence of the Cry2Ah protein, and the mutation mode is that the 354th and 355th amino acids are mutated into serine (S) and proline (P). The mutant Cry2AhM insect-resistant protein has a high insecticidal activity on pests in the order Lepidoptera, and can be used for preparing a fusion insect-resistant protein with high insecticidal activity.

[0008] The mutant can be directly synthesized by using a coding gene, or obtained by a conventional PCR amplification method.

[0009] The application provides a coding gene of the mutant Cry2AhM insect-resistant protein, and the sequence of the coding gene is shown as SEQ ID No. 2. The coding gene is obtained by optimizing the coding sequence of the Cry2AhM insect-resistant protein according to the codons of plant genes such as cotton, and the A+T content in the optimized coding sequence is reduced from 66.09% to 61.45%, and the G+C content is increased from 33.91% to 38.55%.

[0010] The application provides a fusion insect-resistant protein FP2Ah1Ac, which is composed of the mutant Cry2AhM insect-resistant protein and F2A protein and GFM Cry1A protein.

[0011] The F2A is a self-cleaving peptide, and the GFM Cry1A protein is disclosed in a patent with the application number CN95119563.8. Since the action mechanisms of the GFM Cry1A protein and the mutant Cry2AhM insect-resistant protein are different, the fusion insect-resistant protein FP2Ah1Ac has a large insecticidal spectrum, can simultaneously act on multiple pests or different development stages of the same pest, and thus can efficiently kill pests in the order Lepidoptera. Experiments prove that the transgenic cotton expressing the fusion insect-resistant protein FP2Ah1Ac has excellent cotton bollworm resistance, and the corrected mortality of cotton bollworms fed with leaves of the transgenic cotton for 5 days reaches more than 99%. Meanwhile, the normal growth of the plants is not affected.

[0012] Preferably, the amino acid sequence of the fusion insect-resistant protein FP2Ah1Ac is shown as SEQ ID No. 3.

[0013] The application provides a fusion insect-resistant gene, and the fusion insect-resistant gene codes the fusion insect-resistant protein FP2Ah1Ac, and the sequence of the fusion insect-resistant gene is shown as SEQ ID No. 4.

[0014] The fusion pest-resistant gene is a 3.774 kb new pest-resistant gene formed by fusing the optimized coding sequence (i.e., the sequence shown in SEQ ID No. 2) of the pest-resistant protein mutant Cry2AhM and the coding sequence (1803 bp) of the GFM Cry1A protein through the F2A self-cleavage peptide coding sequence (72 bp). The fusion pest-resistant gene can highly express the fusion pest-resistant protein FP2Ah1Ac with the amino acid sequence shown in SEQ ID No. 3 in plants.

[0015] The fifth aspect of the present application provides the use of the above-mentioned fusion pest-resistant gene in constructing an expression vector, an expression cassette, a transgenic cell line or a plant expressing the above-mentioned fusion pest-resistant protein FP2Ah1Ac.

[0016] Preferably, the backbone vector of the expression vector is a pBI21 plasmid or a PUC19 plasmid.

[0017] Preferably, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0018] Further preferably, the plant includes cotton, corn, rice, tobacco, wheat, rape and alfalfa.

[0019] The sixth aspect of the present application provides a plant expression vector pGBI-FP2Ah1Ac, and a construction method thereof specifically includes the following steps: S1, cloning the above-mentioned fusion pest-resistant gene into a PUC19 plasmid through double enzyme digestion, then connecting a 35S promoter to the 5' end of the fusion pest-resistant gene, and cloning a T-nos terminator to the 3' end of the fusion pest-resistant gene to construct an intermediate vector PUC19-P35-FP2Ah1Ac-TNOS; S2, connecting the intermediate vector PUC19-P35-FP2Ah1Ac-TNOS to a pBI121 plasmid to obtain the plant expression vector pGBI-FP2Ah1Ac.

[0020] Preferably, in S1, the fusion pest-resistant gene is cloned into the PUC19 plasmid through double enzyme digestion of Bam H I and Xho I.

[0021] Preferably, in S1, the 35S promoter is connected to the 5' end of the fusion pest-resistant gene through double enzyme digestion of Hin d Ⅲ and Bam H I.

[0022] Preferably, in S1, the T-nos terminator is cloned to the 3' end of the fusion pest-resistant gene through double enzyme digestion of Xho I and Eco R I.

[0023] Preferably, in S2, the intermediate vector PUC19-P35-FP2Ah1Ac-TNOS is connected to the pBI121 plasmid by double enzyme digestion of RI and d III. Hin d Ⅲ and Eco RI double enzyme digestion connects the intermediate vector PUC19-P35-FP2Ah1Ac-TNOS to the pBI121 plasmid.

[0024] The seventh aspect of the present application provides the above-mentioned fusion pest-resistant protein FP2Ah1Ac, fusion pest-resistant gene FP2Ah1Ac and the application of the plant expression vector pGBI-FP2Ah1Ac in cultivating pest-resistant transgenic plants.

[0025] Preferably, the plants include cotton, corn, rice, tobacco, wheat, rape and alfalfa.

[0026] The eighth aspect of the present application provides a method for cultivating pest-resistant transgenic plants, which specifically comprises the following steps: SI, introducing an expression vector expressing the above-mentioned fusion pest-resistant protein FP2Ah1Ac into a target plant to obtain a transgenic plant synchronously expressing the pest-resistant protein mutant Cry2AhM and the GFM Cry1A protein; SII, screening a transgenic plant with strong pest resistance from the transgenic plant obtained in SI, thereby obtaining the pest-resistant transgenic plant.

[0027] Preferably, the plants are dicotyledonous plants or monocotyledonous plants.

[0028] Further preferably, the plants include cotton, corn, rice, tobacco, wheat, rape and alfalfa.

[0029] More preferably, the plants are cotton.

[0030] Illustratively, the cotton can be selected from the land cotton line Jin668.

[0031] Preferably, the expression vector is an expression vector containing the above-mentioned fusion pest-resistant gene.

[0032] Preferably, the expression vector is the above-mentioned plant expression vector pGBI-FP2Ah1Ac.

[0033] Preferably, the method for introducing the expression vector into the target plant in SI is Agrobacterium transformation.

[0034] The application has the advantages that: (1) the fusion pest-resistant protein provided by the application has good insecticidal effect on cotton bollworm, and the insecticidal efficiency of the plant leaves can reach 99% after the cotton is introduced, and the normal growth of the plant is not affected; (2) the fusion pest-resistant gene of the application is fused by two different source insecticidal genes, which avoids the problem of weakening or loss of pest resistance due to single pest-resistant gene; (3) the expression vector provided by the application, in particular the plant expression vector pGBI-FP2Ah1Ac, can make the recipient cotton obtain high resistance to cotton bollworm, which has important significance for reducing the damage of cotton bollworm and other lepidopteran pests in the cotton production process, reducing the use of pesticides, reducing the production cost and improving the cotton yield. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the plasmid map of the plant expression vector pGBI-FP2Ah1Ac in the embodiment 3 of the application; Figure 2 It is the electrophoresis map of PCR identification of the transgenic cotton in the embodiment 4 of the application; 1# lane is a positive control, 2# lane is a negative control, 3#-6# lanes are the PCR electrophoresis results of four T0 generation transgenic single plants (FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, FP2Ah1Ac-4) obtained by amplification of CryFP / RP primers respectively; Figure 3 It is the identification photo of Bt protein test strip of the transgenic cotton in the embodiment 4 of the application; 1 is the identification result of non-transgenic cotton Jin668, 2-5 are respectively the identification results of transgenic plants FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, FP2Ah1Ac-4; Figure 4 It is the photo of the leaf insecticidal efficiency detection effect comparison test of non-transgenic cotton and transgenic cotton in the test example of the application; wherein WT is non-transgenic cotton Jin668. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the application more clear and obvious, the application will be further described in detail below in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the application, and are not used to limit the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0037] By introducing Bt gene into plants, specific insect resistance can be conferred to plants. Currently, 8 insect resistance genes have been introduced into cotton and have been commercialized. However, with the large-scale popularization of transgenic insect-resistant crops, pests gradually develop resistance to Bt proteins. For example, the insect resistance activity of Cry2Ah protein to the cotton bollworm and other pests that threaten cotton production at the present stage is not ideal.

[0038] The embodiment of the present application provides an insect-resistant protein mutant Cry2AhM, and the amino acid sequence of the insect-resistant protein mutant Cry2AhM is shown as SEQ ID No. 1. The insect-resistant protein mutant Cry2AhM has high insecticidal activity on lepidopteran pests, and can be used for preparing a fusion insect-resistant protein with high insecticidal activity.

[0039] The embodiment of the present application also provides a coding gene of the insect-resistant protein mutant Cry2AhM, and the sequence is shown as SEQ ID No. 2.

[0040] Based on the insect-resistant protein mutant Cry2AhM, the embodiment of the present application also provides a fusion insect-resistant protein FP2Ah1Ac which is composed of the insect-resistant protein mutant Cry2AhM, F2A protein and GFM Cry1A protein.

[0041] The embodiment of the present application also provides a fusion insect-resistant gene for coding the fusion insect-resistant protein FP2Ah1Ac, and the sequence is shown as SEQ ID No. 4.

[0042] The embodiment of the present application also provides a plant expression vector pGBI-FP2Ah1Ac, and the plant expression vector pGBI-FP2Ah1Ac is constructed by using the fusion insect-resistant gene, PUC19 plasmid and pBI121 plasmid.

[0043] The embodiment of the present application also provides the application of the fusion insect-resistant protein FP2Ah1Ac, the fusion insect-resistant gene and the plant expression vector pGBI-FP2Ah1Ac in cultivating insect-resistant transgenic plants, and provides a method for cultivating insect-resistant transgenic plants. FP2Ah1Ac

[0044] The scheme of the present application is described through specific embodiments as follows.

[0045] In the following embodiments, other reagents or instruments are commercially available general products without special instructions. In the following embodiments, the methods used are general methods in the art without special instructions.

[0046] Embodiment 1 The embodiment provides an insect-resistant protein mutant Cry2AhM, and the amino acid sequence of the insect-resistant protein mutant Cry2AhM is shown as SEQ ID No. 1. ​

[0047] The insect-resistant protein mutant Cry2AhM was synthesized directly using the coding gene shown in SEQ ID No. 2.

[0048] Example 2 This embodiment provides a fusion insect-resistant protein FP2Ah1Ac and its encoding gene. The amino acid sequence of the fusion insect-resistant protein FP2Ah1Ac is shown in SEQ ID No. 3, and the nucleotide sequence of its encoding gene is shown in SEQ ID No. 4.

[0049] The coding gene, as shown in SEQ ID No. 4, was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd. 1–1899 bp encodes the insect-resistant protein mutant Cry2AhM, 1900–1971 bp encodes the F2A self-splicing peptide, and 1972–3774 bp encodes the GFM Cry1A protein. Subsequently, the fusion insect-resistant protein FP2Ah1Ac was synthesized using this coding gene.

[0050] Example 3 This embodiment provides a plant expression vector pGBI-FP2Ah1Ac, the specific construction method of which is as follows: (1) Through Bam HI and Xho I. Double enzyme digestion was used to clone the fusion insect-resistant gene, with the nucleotide sequence shown in SEQ ID No. 4, into the PUC19 plasmid. Then, using... Hin dⅢ and Bam HI double digestion was performed, and the 1152 bp 35S promoter was ligated to the 5' end of the fused insect-resistant gene. Finally, [the process was] utilized... Xho I and Eco The RI double enzyme digestion was used to clone the 518bp T-nos terminator into the 3' end of the fusion insect-resistant gene, and the intermediate vector PUC19-P35-FP2Ah1Ac-TNOS was constructed.

[0051] (2) Utilization Hin dⅢ and Eco The intermediate vector PUC19-P35-FP2Ah1Ac-TNOS was ligated into the pBI121 plasmid by double digestion with RI enzymes, resulting in the plant expression vector pGBI-FP2Ah1Ac containing the above-mentioned fused insect-resistant gene (plasmid map shown). Figure 1 (As shown).

[0052] Example 4 This embodiment provides a method for cultivating insect-resistant transgenic cotton using the plant expression vector pGBI-FP2Ah1Ac constructed in Example 3. The specific steps are as follows: (1) Transformation of Agrobacterium with plant expression vector pGBI-FP2Ah1Ac Mix 2-5 pg of plant expression vector pGBI-FP2Ah1Ac with 100 pL of Agrobacterium GV3101 competent cells, and place on ice for 5 min. Then add the mixture to a 2-mm electroporation cuvette, and perform electroporation at 2500 V. After electroporation, quickly add 800 pL of LB liquid medium to the cuvette, and transfer to a 1.5-mL centrifuge tube. Incubate at 28°C and 180 rpm for 5 h. Take 100 pL of the bacterial solution, and spread on LB solid medium containing kanamycin and rifampicin (both at a concentration of 50 pg / mL), and incubate at 28°C for 48 h. Randomly pick 5 resistant clones, and send to GenScript Biotech (Shanghai) Co., Ltd. for sequencing verification. Select one monoclonal bacteria with correct expression vector sequence.

[0053] (2) Agrobacterium-mediated genetic transformation of cotton and obtaining of transgenic plants Use the transformed Agrobacterium obtained in step (1) to infect the cotton hypocotyls, and then obtain transgenic plants through tissue culture. The specific steps are as follows: (a) Sterile seedling preparation: Surface sterilize the seeds of Jin668 with 70% ethanol for 30 s, and then soak in 30% H2O2 for 2 h. Then rinse with sterile distilled water three times to remove the residual H2O2 on the surface of the seeds. Soak the sterilized seeds in sterile water at 28°C overnight. After the seeds germinate and the seed coat is removed, sow on 1 / 2 Murashige & Skoog (MS) medium. After sowing, incubate the seeds at 28°C in the dark for 4 days, and then incubate under 16 h light / 8 h dark conditions for 2 days. Cut the hypocotyls of the seedlings into 0.5-cm lengths, and use as explants for callus induction.

[0054] (b) Genetic transformation and obtaining of regenerated plants: Inoculate the Agrobacterium containing the plant expression vector pGBI-FP2Ah1Ac obtained in step (1) into 100 mL of LB liquid medium (containing 50 mg / L of kanamycin and rifampicin), and incubate at 28°C and 250 rpm for 8-10 h. Then centrifuge at 4000 rpm for 10 min, collect the cultured Agrobacterium cells, and resuspend in MS liquid medium to OD 600For 0.4, the step (a) divided explants were soaked in resuspension solution for 30 min. Then, the infected explants were dried on sterile filter paper and transferred to co-culture medium (MSB5 solid medium containing 0.05 mg / L KT, 2.5 mg / L 2,4-D), and co-cultured for 2 days at 24°C in the dark. The infected explants were transferred to CIM callus induction medium (MSB5 solid medium containing 100 mg / L kanamycin, 500 mg / L cefotaxime, 0.05 mg / L KT, 2.5 mg / L 2,4-D), and cultured for 2-3 months at 28°C under 16 h light / 8 h dark conditions to induce callus.

[0055] The actively growing callus was selected and transferred to EIM embryo induction medium (MSB5 solid medium containing 100 mg / L kanamycin, 500 mg / L cefotaxime), and cultured for 2-3 months at 28°C under 16 h light / 8 h dark conditions. Then, the green healthy callus tissue was transferred to EIM without kanamycin to induce embryo development and form seedlings. Four seedlings with normal appearance were selected and transferred to MS medium containing 200 mg / L IAA, and cultured at 28°C under 16 h light / 8 h dark conditions to induce rooting, and finally developed into transgenic regenerated plants.

[0056] (3) PCR identification of transgenic plants The four transgenic plants obtained were transplanted in the field, and each plant grew well in the field and could grow normally. The genomic DNA of each plant was extracted, and then specific primers (CryFp / RP) designed for the fusion insect-resistant protein FP2Ah1Ac encoding gene were used for PCR amplification, and the product size was 561 bp (see Figure 2 ). The electrophoresis analysis results of the PCR product showed that the characteristic band of the fusion insect-resistant gene was amplified in the four T0 generation transgenic plants (FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, FP2Ah1Ac-4). The identification primers of the fusion gene are: CryFP: 5'-GCTTCTAACGTTAATACAACCAC-3' (SEQ ID No. 5); CryRP: 5'-GTAGAGATTGCTCAATCCTTCCAAC-3' (SEQ ID No. 6).

[0057] PCR reaction system (50 μL): 2x Tag Mix 25 μL, primer 1 (10 mM) 1 μL, primer 2 (10 mM) 1 μL, ddH2O 23 μL. PCR reaction conditions: 98°C 2 min; 98°C 2 min, 60°C 30 s, 72°C 30 s, 30 cycles; 72°C 5 min.

[0058] The leaves of the transgenic plants were detected by CrylAc test strips, and the results (as shown in Figure 3 ) showed that the four transgenic plants all expressed insecticidal proteins.

[0059] The above results show that the four transgenic cottons containing the fusion insect-resistant gene were obtained by using the plant expression vector pGBI-FP2Ah1Ac in the present example.

[0060] Test Example In the present test example, the insect resistance of the transgenic cotton obtained in Example 4 was identified.

[0061] (1) The T3 generation homozygous transgenic cotton leaves of FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, and FP2Ah1Ac-4 in Example 4 were collected, cut into 2 cm x 2 cm squares, and placed in culture dishes.

[0062] (2) Five 1-day-old newly hatched cotton bollworms were fed in each culture dish. The leaves of wild-type Jin668 plants grown at the same time were used as negative controls. After 5 days of feeding, the mortality rate and leaf damage were investigated.

[0063] The results are shown in Table 1. The corrected mortality rate of the cotton bollworms fed with the transgenic leaves reached more than 99%.

[0064] Leaf surface integrity observation found (as shown in Figure 4 ), the non-transgenic leaves were severely damaged with a large number of insect holes; while the transgenic cotton leaves had only a small number of insect holes and were basically intact, further proving that the FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, and FP2Ah1Ac-4 transgenic cottons had strong insect resistance.

[0065] Table 1: Insecticidal efficiency detection of transgenic cotton

[0066] As can be seen from the above examples, the FP2Ah1Ac-1, FP2Ah1Ac-2, FP2Ah1Ac-3, and FP2Ah1Ac-4 transgenic cottons obtained by introducing the fusion insect-resistant gene of the present application into cotton all showed strong activity against cotton bollworms.

[0067] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement or improvement made in the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A mutant insect-resistant protein, Cry2AhM, characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

2. The encoding gene of the insect-resistant protein mutant Cry2AhM according to claim 1, characterized in that, Its sequence is shown in SEQ ID No.

2.

3. A fusion of insect-resistant protein FP2Ah1Ac, characterized in that, It is composed of the insect-resistant protein mutant Cry2AhM as described in claim 1, and the fusion of F2A protein and GFM Cry1A protein.

4. The fusion insect-resistant protein FP2Ah1Ac according to claim 3, characterized in that, Its amino acid sequence is shown in SEQ ID No.

3.

5. A fusion of insect-resistant genes, characterized in that, The fusion insect-resistant gene encodes the fusion insect-resistant protein FP2Ah1Ac of claim 4, the sequence of which is shown in SEQ ID No.

4.

6. The use of the fusion insect-resistant gene of claim 5 in constructing expression vectors, expression cassettes, transgenic cell lines or plants expressing the fusion insect-resistant protein FP2Ah1Ac of claim 3 or 4.

7. The application according to claim 6, characterized in that, The backbone vector of the expression vector is pBI21 plasmid and / or PUC19 plasmid; and / or The plant is a dicotyledonous plant or a monocotyledonous plant.

8. A plant expression vector pGBI-FP2Ah1Ac, characterized in that, Its construction method specifically includes the following steps: S1. The fusion insect-resistant gene described in claim 5 is cloned into the PUC19 plasmid by double enzyme digestion. Then, the 35S promoter is linked to the 5' end of the fusion insect-resistant gene, and the T-nos terminator is cloned to the 3' end of the fusion insect-resistant gene to construct the intermediate vector PUC19-P35-FP2Ah1Ac-TNOS. S2. The intermediate vector PUC19-P35-FP2Ah1Ac-TNOS is linked to the pBI121 plasmid to obtain the plant expression vector pGBI-FP2Ah1Ac.

9. The application of the fusion insect-resistant protein FP2Ah1Ac according to claim 3 or 4, the fusion insect-resistant gene according to claim 5, or the plant expression vector pGBI-FP2Ah1Ac according to claim 8 in the cultivation of insect-resistant transgenic plants.

10. A method for cultivating insect-resistant transgenic plants, characterized in that, Specifically, the steps include the following: S1. Introduce the expression vector expressing the insect-resistant protein FP2Ah1Ac as described in claim 3 or 4 into the target plant to obtain a transgenic plant that simultaneously expresses the insect-resistant protein mutant Cry2AhM and GFM Cry1A protein; SII. Select transgenic plants with strong insect resistance from the transgenic plants obtained from SI to obtain the insect-resistant transgenic plants.

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