Expression vector containing insect-resistant and glyphosate-resistant herbicide gene and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly discloses an insect-resistant gene and an insect-resistant fusion protein coded by the insect-resistant gene, and the nucleotide sequence of the insect-resistant gene is shown as SEQ ID NO.2. The invention also discloses an expression vector containing the insect-resistant gene and the glyphosate herbicide-resistant gene, wherein the glyphosate herbicide-resistant gene comprises a GR79EPSPS gene and a GAT gene. The insect-resistant gene is formed by fusing insect-resistant genes from different sources, so that not only is the insect resistance high, but also the problem that the resistance is easy to lose due to single resistance is avoided; secondly, the insect-resistant gene disclosed by the invention is optimized, so that the expression efficiency of the insect-resistant gene in plants is high; besides, the expression vector disclosed by the invention can enable plants to obtain insect-resistant and glyphosate herbicide-resistant characters at the same time, is simple in transformation and high in transformation efficiency, not only solves insect damage and weed damage at the same time in production, but also greatly reduces labor input and production cost, and has important significance for improving yield and quality and improving economic benefits.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an expression vector containing insect-resistant and glyphosate-resistant herbicide genes, and the use of the expression vector; it also relates to an insect-resistant fusion protein. Background Art

[0002] Bt proteins can specifically kill a wide range of agricultural pests, including Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Trichophagous, while being harmless to non-target insects, birds, humans, and livestock. Introducing Bt genes into plants can specifically control major pests and has significant economic value. The first commercialized insect-resistant crops internationally were Monsanto's Cry1Ac transgenic cotton and Cry1Ab transgenic corn. my country first introduced the synthetic GFM Cry1A gene into cotton in 1992, producing the first generation of domestically produced single-value insect-resistant cotton and beginning industrial application. However, with the widespread deployment of insect-resistant cotton, Lepidoptera pests such as the cotton bollworm developed resistance to it, resulting in the ineffectiveness of crops like Bt cotton in killing pests (Tabashnik et al., Nature Biotechnology, 2008). This suggests that the use of a single insect-resistant gene can easily lead to pest resistance.

[0003] Liao et al. (Journal of Invertebrate Pathology, 2002), through functional studies of the Bt insecticidal protein family, found that different Bt proteins exhibit varying mechanisms of killing the same pest, making it difficult for pests to develop cross-resistance to two proteins. Therefore, co-expressing two or more Bt proteins not only enhances the insecticidal activity of transgenic insect-resistant crops but also prevents pests from developing resistance to them. Therefore, introducing two or more insect-resistant genes into crops is an effective way to prevent pests from developing resistance to transgenic insect-resistant crops.

[0004] Glyphosate is a broad-spectrum, systemic herbicide widely used for field weed control on crops such as corn, soybeans, and cotton, as well as in orchards and rubber plantations. It is one of the most widely used herbicides worldwide. However, glyphosate is also a non-selective, lethal herbicide, and direct field spraying can harm crops. The discovery of the glyphosate-resistant EPSPS gene and the widespread use of glyphosate-resistant transgenic crops have addressed the issue of glyphosate's harmful effects on crops. Guo Sandui et al. (see Patent ZL2014102047036) simultaneously introduced the GR79 EPSPS gene and the GAT gene into cotton, creating transgenic cotton resistant to four times the concentration of glyphosate used in production. Currently, the transgenic cotton line GGK2 containing the GR79 EPSPS and GAT genes has obtained the China Genetically Modified Organism Production and Application Certificate and has begun industrial application. Liang Chengzhen et al. (Plant Biotechnology Journal. 2017) disclosed that the combined application of GR79 EPSPS gene and GAT gene can be used to cultivate cotton with low residue and high resistance to glyphosate.

[0005] The construction of co-expression vectors containing multiple genes, such as insect-resistant genes and herbicide-tolerant genes, allows for the simultaneous introduction of both genes into cotton. This approach can simultaneously address the challenges of bollworm and weed damage in cotton production, while also improving transgenic efficiency. Currently, with the exception of commercially available transgenic corn that possesses both insect-resistant and herbicide-tolerant traits, other transgenic crops, such as cotton and soybeans, only possess either insect-resistant or herbicide-tolerant traits, failing to address both pest and weed damage simultaneously. Therefore, the development of new co-expression vectors containing both insect-resistant and herbicide-tolerant genes is urgently needed. Summary of the Invention

[0006] In order to solve the problem that a single insect-resistant gene easily leads to loss of resistance, the present invention aims to provide an insect-resistant fusion protein.

[0007] The present invention provides an insect-resistant fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] The present invention also provides an insect-resistant gene encoding the above-mentioned insect-resistant fusion protein, which is named Gmf Cry1A. The nucleotide sequence of the insect-resistant gene is shown in SEQ ID NO.2.

[0009] The insect-resistant gene of the present invention is formed by fusing the first and second structural domains of Cry1Ab with the third structural domain of Cry1Ac.

[0010] The present invention also provides an expression vector, an expression cassette, a transgenic cell line or a plant containing the insect-resistant gene.

[0011] The present invention also provides the application of the insect-resistant gene in cultivating insect-resistant transgenic plants.

[0012] The present invention also provides the use of the above-mentioned expression vector, expression cassette and transgenic cell line in cultivating insect-resistant transgenic plants.

[0013] In the above applications, the plants refer to cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa, but are not limited to the listed plants.

[0014] The present invention also provides an expression vector containing the insect-resistant gene and the glyphosate-resistant herbicide gene.

[0015] In the above expression vector, the nucleotide sequence of the insect-resistant gene is shown in SEQ ID NO.2.

[0016] In the above expression vector, the glyphosate herbicide-resistant genes include the GR79EPSPS gene and the GAT gene.

[0017] The nucleotide sequence of the GR79EPSPS gene is shown in SEQ ID NO. 3. The nucleotide sequence of the GAT gene is shown in SEQ ID NO. 4.

[0018] In the above-mentioned expression vector, the promoter of the insect-resistant gene is the cotton seed globulin gene promoter pGhαGLOA (p alpha globulin), and the nucleotide sequence of the cotton seed globulin gene promoter pGhαGLOA is as shown in sequence 1 in the sequence table of the patent "Cloning and Functional Identification of Upland Cotton Seed Globulin Gene Promoter" (patent number: ZL2015105075070).

[0019] In the above expression vectors, the vectors refer to: pBI21, PUC19, etc.

[0020] The present invention also provides the use of the above expression vector in cultivating insect-resistant and glyphosate-resistant plants.

[0021] The plants mentioned include cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa, but are not limited to the listed plants.

[0022] The present invention also provides an expression cassette, a transgenic cell line or a plant containing the above expression vector.

[0023] The present invention also provides a method for preparing the above expression vector, comprising the following steps:

[0024] (1) The cotton seed globulin promoter GhαGLOA (p_alpha_globulin) was cloned into the PUC19 vector by double digestion with Hind III and BamH I;

[0025] (2) using BamH I and Pst I to connect the Ω and Cozak enhancer sequences to the 3' end of the promoter described in step (1);

[0026] (3) connecting the artificially synthesized insect-resistant gene Gmf Cry1A (whose nucleotide sequence is shown in SEQ ID No: 2) to the 3' end of the enhancer sequence described in step (2) via Pst I and Xho I;

[0027] (4) Poly A and T NOS in the expression vector pGBIGRGAT were cloned into the 3' end of the Gmf Cry1A gene by double digestion with Pme I and Xho I to obtain an intermediate expression vector;

[0028] (5) Obtaining the Gmf Cry1A gene expression cassette by double digesting the intermediate expression vector obtained in step (4) with Pme I and Hind III;

[0029] (6) The Gmf Cry1A gene expression cassette obtained in step (5) was connected to pGBIGRGAT double-digested with Pme I and Hind III to obtain an expression vector containing the insect-resistant and glyphosate-resistant herbicide genes.

[0030] In the above preparation method, the nucleotide sequence of the Ω enhancer sequence in step (2) is shown as SEQ ID NO.5.

[0031] In the above preparation method, the nucleotide sequence of the Cozak enhancer sequence in step (2) is shown as SEQ ID NO.6.

[0032] In the above preparation method, the expression vector pGBIGRGAT described in step (6) refers to a bivalent recombinant expression vector carrying the GR79EPSPS gene and the GAT gene; the composition of the bivalent recombinant expression vector pGBIGRGAT and its construction method are shown in Example 2 in the specification of the patent "An expression vector containing a glyphosate-resistant gene and its application" (patent number: ZL2014102047036).

[0033] The present invention also provides a method for cultivating insect-resistant and glyphosate-resistant transgenic plants, comprising the following steps:

[0034] (1) introducing the above expression vector into a target plant to obtain a transgenic plant that simultaneously expresses an insect-resistant gene and a glyphosate-tolerant gene;

[0035] (2) Screening transgenic plants with enhanced insect resistance and glyphosate resistance from the transgenic plants obtained in step (1).

[0036] In the above application or method, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0037] In the above applications, the monocotyledonous and dicotyledonous plants include cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa, but are not limited to the listed plants.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The insect-resistant fusion protein or insect-resistant gene of the present invention has a good insecticidal effect on cotton bollworms. After being introduced into cotton, the insecticidal efficiency of the plant leaves can reach 99%. (2) The insect-resistant gene of the present invention is formed by the fusion of insecticidal genes from different sources, which avoids the problem of weakening or loss of insect resistance due to the single insect-resistant gene, that is, it is not easy for pests such as cotton bollworms to develop resistance to it, and the insect-resistant trait is stable and long-lasting. (3) The insect-resistant gene of the present invention is optimized according to the codon preference of plants and has high expression efficiency. (4) The expression vector provided by the present invention can enable the recipient cotton to obtain dual resistance traits of insect resistance and glyphosate herbicide resistance at the same time. The transformation is simple and the transformation efficiency is high, which is of great significance for reducing the damage caused by lepidopteran pests such as cotton bollworms and weeds in the cotton production process, reducing labor input, reducing production costs and increasing cotton yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 . Electrophoresis patterns of transgenic cotton plants of the present invention identified by PCR using GMFCry1A-FP / GMFCry1A-RP, GR79EPSPS-FP / GR79EPSPS-RP and GAT-FP / GAT-RP as primers, respectively; wherein lanes 1#, 2#, and 3# are the products of negative control PCR amplification using the above three primer pairs; lanes 4#, 5#, and 6# are GBS1; lanes 7#, 8#, and 9# are GBS3; lanes 10#, 11#, and 12# are GBS4; and lanes 13#, 14#, and 15# are GBS8-2.

[0040] Figure 2 Comparative photos of transgenic cotton plants of the present invention identified using herbicide-tolerant protein test strips; 1 is GBS1; 2 is GBS3; 3 is GBS4; 4 is GBS8-2.

[0041] Figure 3 Comparative photos of transgenic cotton plants of the present invention identified using insect-resistant protein test strips; 1 is GBS1; 2 is GBS3; 3 is GBS4; 4 is GBS8-2.

[0042] Figure 4 Comparative photos of the field test of the transgenic cotton GBS8-2 resistant to glyphosate herbicide of the present invention; GBS8-2 is the insect-resistant and glyphosate-resistant transgenic cotton of the present invention; WT is the non-transgenic recipient cotton.

[0043] Figure 5 Comparative photos of leaves of the transgenic cotton GBS8-2 of the present invention in the field insect resistance test; GBS8-2 is the insect-resistant and glyphosate-resistant transgenic cotton of the present invention; WT is the non-transgenic recipient cotton. DETAILED DESCRIPTION

[0044] The present invention is further described below by means of specific examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0045] Example 1 Design and artificial synthesis of insect-resistant genes of the present invention

[0046] Based on the Bacillus thuringiensis Cry1Ab and Cry1Ac protein sequences published in the NCBI database, the coding sequences for the first and second domains of Cry1Ab and the third domain of Cry1Ac were extracted, respectively. The two incomplete coding sequences were fused to form a new insect-resistant gene of 3516 bp. Based on this, the original insect-resistant gene sequence was optimized based on cotton codon preference. While maintaining the amino acid sequence of the encoded protein, some bacterial-preferred codons were replaced with cotton-preferred codons. Compared to the original nucleotide sequence, the G / C content of the optimized gene sequence increased from 34.22% to 43.46%. The nucleotide sequence of the optimized insect-resistant gene is shown in SEQ ID NO. 2, and the gene is named Gmf Cry1A. The amino acid sequence of the insect-resistant fusion protein encoded by this gene is shown in SEQ ID NO. 1.

[0047] Example 2 Construction of the Expression Vector Containing Insect-Resistant and Glyphosate-Tolerant Genes of the Present Invention

[0048] Proceed as follows:

[0049] (1) The cotton seed globulin core promoter GhαGLOA (p_alpha_globulin (specific nucleotide sequence see patent "Cloning and Functional Identification of Upland Cotton Seed Globulin Gene Promoter" (Patent No.: ZL2015105075070) sequence table sequence 1) was cloned into the PUC19 vector by double enzyme digestion with Hind III and BamH I, and the Ω and Cozak enhancer sequences were connected to the 3' end of the above promoter by BamH I and Pst I; then the artificially synthesized Gmf Cry1A gene (the nucleotide sequence of the insect-resistant gene is shown in SEQ ID NO.2, chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd.) was connected to the 3' end of the enhancer sequence by Pst I and Xho I; finally, the Poly A and T NOS in the expression vector pGBIGRGAT were cloned into the Gmf The 3' end of the Cry1A gene was obtained; an intermediate expression vector containing the insect-resistant gene GmfCry1A was obtained, and the intermediate expression vector was named: PUC19-p alpha globulin-GMFCry1A-TNOS.

[0050] (2) The intermediate expression vector PUC19-p alphaglobulin-GMFCry1A-TNOS obtained in step (1) was digested with PmeI and HindIII to obtain a Gmf Cry1A gene expression cassette. At the same time, the obtained Gmf Cry1A gene expression cassette was connected to the PmeI and HindIII double-enzyme-digested bivalent herbicide-resistant gene expression vector pGBIGRGAT (constructed according to the method described in Example 2 of the patent "An Expression Vector Containing a Glyphosate-Resistant Gene and Its Application" (Patent No.: ZL2014102047036)) to obtain a plant expression vector containing insect-resistant and glyphosate-resistant genes. The expression vector was named: pGBI-Gmf Cry1A-GR79EPSPS-GAT.

[0051] Example 3 The experiment of obtaining the insect-resistant and herbicide-tolerant transgenic cotton of the present invention by using the transgenic method was carried out as follows:

[0052] (1) Plant expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT was used to transform Agrobacterium

[0053] 2-5 μg of the plant expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT obtained in Example 2 was mixed with 100 μL of competent Agrobacterium GV3101 cells and placed on ice for 5 minutes. The mixture was then added to a 2 mm electroporation cuvette and electroporated at 2500V. After electroporation, 800 μL of LB liquid medium was quickly added and the cells were transferred to a 1.5 mL centrifuge tube. The cells were activated and cultured at 28°C and 180 rpm for 5 hours. 100 μL of the bacterial solution was spread onto a plate containing LB solid medium containing kanamycin and rifampicin (both at a concentration of 50 μg / mL) and incubated at 28°C for 48 hours. Five resistant clones were randomly selected and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The Agrobacterium containing the correct expression vector sequence was selected, namely, the Agrobacterium containing the expression vector pGBI-GmfCry1A-GR79EPSPS-GAT.

[0054] (2) Agrobacterium-mediated genetic transformation of cotton and acquisition of transgenic plants

[0055] Using upland cotton line Jin668 (with a deposit number of CCTCC NO: P201519, a cotton line bred by Huazhong Agricultural University) as a recipient, the transformed Agrobacterium obtained in step (1) was used to infect cotton hypocotyls, and then transgenic plants were obtained by tissue culture. The specific steps are as follows:

[0056] (a) Sterile seedling preparation: Jin668 seeds were surface-sterilized with 70% ethanol for 30 seconds, then immersed in 30% H2O2 for 2 hours, and then rinsed three times with sterile distilled water to remove any residual H2O2 on the seed surface. The sterilized seeds were soaked in sterile water at 28°C overnight. After the seeds germinated and turned white, the seed coats were removed and the seeds were sown on 1 / 2 Murashige & Skoog (MS) medium. The sown seeds were cultured at 28°C in the dark for 4 days, followed by 16 hours of light / 8 hours of darkness for 2 days. The hypocotyls of the seedlings were cut and divided into 0.5 cm lengths to serve as explants for callus induction.

[0057] (b) Genetic transformation and regeneration of plants: The Agrobacterium containing the expression vector pGBI-Gmf Cry1A-GR79EPSPS-GAT obtained in step (1) was inoculated into 100 mL of LB liquid medium (containing 50 mg / L glyphosate herbicide and rifampicin) and cultured at 28°C and 250 rpm for 8 to 10 hours. The cultured Agrobacterium cells were then collected by centrifugation at 4000 rpm for 10 minutes and resuspended in MS liquid medium to an OD of 0. 600The explants separated in step (a) were immersed in the resuspension solution at a concentration of 0.4. The infected explants were then dried on sterile filter paper and transferred to co-cultivation medium (MSB5 solid medium containing 0.05 mg / L KT and 2.5 mg / L 2,4-D) 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 glyphosate herbicide, 500 mg / L cephalosporin, 0.05 mg / L KT, and 2.5 mg / L 2,4-D) and cultured at 28°C under 16 hours of light / 8 hours of darkness for 2-3 months to induce callus cells.

[0058] (c) Actively growing calli are selected and transferred to EIM embryo induction medium (MSB5 solid medium containing 100 mg / L glyphosate and 500 mg / L cephalosporin) and cultured at 28°C under 16-hour light / 8-hour dark conditions for 2-3 months. Green, healthy calli are then transferred to EIM without kanamycin to induce embryogenesis and form shoots. Shoots that appear to be developing normally are selected and transferred to MS medium containing 200 mg / L IAA. Rooting is induced at 28°C under 16-hour light / 8-hour dark conditions, ultimately resulting in transgenic regenerated plants.

[0059] (3) PCR identification of transgenic plants

[0060] The transgenic plants were transplanted into the field, and genomic DNA was extracted from each plant. PCR amplification and identification were performed using specific primers designed based on the Gmf Cry1A, GR79 EPSPS, and GAT genes. The primers used to identify the Gmf Cry1A gene were:

[0061] GMFCry1A-FP: 5'-CACGGAGGCATAGTCAGCAGG-3' (SEQ ID NO. 7);

[0062] GMFCry1A-RP: 5'-GTGTGCACAGCATTCGTGAG-3' (SEQ ID NO. 8).

[0063] The primers used to identify the GR79 EPSPS gene are:

[0064] GR79EPSPS-FP: 5'-CCTGGAGCAAGGCTACGGAG-3' (SEQ ID NO.9);

[0065] GR79EPSPS-RP: 5'-GGCCTGAGAGGAACTGGCTG-3' (SEQ ID NO. 10).

[0066] The primers used to identify the GAT gene are:

[0067] GAT-FP: 5'-GGACTGCAGCATGATTGATGTG-3' (SEQ ID NO. 11);

[0068] GAT-RP: 5'-GAGGTCCCACAGGAGGAGTG-3' (SEQ ID NO. 12).

[0069] The PCR reaction system (50 μL) consisted of: 2X Tag Mix 25 μL, Primer 1 (10 mM) 1 μL, Primer 2 (10 mM) 1 μL, and ddH2O 23 μL. The PCR reaction conditions were: 98°C for 2 min; 30 cycles of 98°C for 2 min, 58°C for 30 s, and 72°C for 30 s; and 72°C for 5 min.

[0070] Results (see Figure 1 The amplified products of the Gmf Cry1A, GR79 EPSPS, and GAT genes were 524 bp, 546 bp, and 422 bp, respectively. Electrophoresis analysis of the PCR products revealed that characteristic bands for the Gmf Cry1A, GR79 EPSPS, and GAT genes were simultaneously amplified in the four T0 transgenic plants, indicating that four transgenic cotton plants harboring insect-resistant and herbicide-tolerant genes were obtained. These four transgenic plants were designated GBS1, GBS3, GBS4, and GBS8-2.

[0071] (4) The leaves of the transgenic plants were tested using Cry1Ac test strips (purchased from Beijing Aochuang Jinbiao Biological Company) and GR79 EPSPS test strips (from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences). The results (see Figure 2 and Figure 3 ) found that all four transgenic plants simultaneously expressed insecticidal proteins and glyphosate-resistant herbicide proteins.

[0072] Example 4: Test for identification of herbicide tolerance of insect-resistant and herbicide-tolerant transgenic cotton of the present invention

[0073] Proceed as follows:

[0074] (1) The four T0-generation transgenic plants obtained in Example 3 were transplanted into the field and simultaneously sprayed with Roundup glyphosate at a concentration four times the production concentration (8 mg / L). Finally, a single plant was obtained whose growth was not inhibited and whose yield was no different from the control. This plant was designated GBS8-2. Based on this, homozygous transgenic cotton of the T4 generation of GBS8-2 was obtained by screening through multiple generations of continuous glyphosate herbicide spraying.

[0075] (2) GBS8-2 T4 cotton seeds were sown in nutrient pots to cultivate seedlings, yielding a total of 50 seedlings. When the cotton seedlings had three true leaves, they were sprayed with 8 mg / L Roundup glyphosate herbicide. The seedling phenotype was observed 5 days after spraying.

[0076] Results (see Figure 4 All 50 T4 transgenic plants derived from GBS8-2 grew normally, while the wild-type recipient WT plants withered and died. This indicates that the transgenic cotton GBS8-2 obtained in this invention has high tolerance to glyphosate herbicides and has important application value for efficient weed control in cotton production.

[0077] Example 5 Insect resistance identification test of transgenic cotton of the present invention

[0078] (1) Test materials: The T0 generation materials of GBS1, GBS3, GBS4 and GBS8-2 obtained in Example 3 were subjected to several consecutive generations to obtain the corresponding T4 generation materials of GBS1, GBS3, GBS4 and GBS8-2.

[0079] (2) Test methods:

[0080] (1) The T4 generation homozygous transgenic cotton leaves of GBS1, GBS3, GBS4 and GBS8-2 were cut into 2 cm squares and placed in culture dishes.

[0081] (2) Five newly hatched cotton bollworm larvae, fed for one day, were inoculated into each culture dish. Leaves from wild-type plants grown at the same time served as negative controls. Mortality and leaf damage were assessed five days after inoculation.

[0082] Results (see Table 1) showed that the corrected mortality of cotton bollworms fed with the transgenic cotton leaves of the present invention reached over 99%. Figure 5 ), the leaves of the non-transgenic plants (WT) were severely damaged and had many insect holes; while the leaves of the transgenic cotton plants GBS8-2 of the present invention had only a few insect holes and were basically intact, further proving that the transgenic cotton GBS8-2 of the present invention has strong insect resistance.

[0083] Table 1: Test results of insecticidal efficiency of transgenic cotton of the present invention

[0084]

[0085]

[0086] It can be seen from the above examples that the transgenic cotton GBS8-2 of the present invention not only has a strong resistance to glyphosate herbicides, but also has a strong resistance to cotton bollworms. In addition, the insect resistance of the transgenic cotton is stable and is not easily lost.

Claims

1. An insect-resistant fusion protein, characterized in that: The amino acid sequence of the insect-resistant fusion protein is shown in SEQ ID NO.

1.

2. The insect-resistant gene encoding the insect-resistant fusion protein according to claim 1, characterized in that: The nucleotide sequence of the insect-resistant gene is shown in SEQ ID NO.

2.

3. An expression vector, expression cassette, transgenic cell line or plant containing the insect-resistant gene according to claim 2.

4. Use of the insect-resistant fusion protein according to claim 1 or the insect-resistant gene according to claim 2 in cultivating insect-resistant transgenic plants.

5. Use of the expression vector, expression cassette, and transgenic cell line according to claim 3 in cultivating insect-resistant transgenic plants.

6. The use according to claim 4 or 5, characterized in that The plant is cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa.

7. An expression vector containing an insect-resistant gene and a glyphosate-resistant herbicide gene, characterized in that: The expression vector contains the insect-resistant gene and the glyphosate herbicide-resistant gene according to claim 2; wherein the nucleotide sequence of the insect-resistant gene is shown as SEQ ID NO.2; the glyphosate herbicide-resistant gene includes the GR79EPSPS gene and the GAT gene; wherein the nucleotide sequence of the GR79EPSPS gene is shown as SEQ ID NO.3; wherein the nucleotide sequence of the GAT gene is shown as SEQ ID NO.

4.

8. Use of the expression vector according to claim 7 in cultivating insect-resistant and glyphosate-resistant plants; characterized in that: The plants are cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa.

9. An expression cassette, a transgenic cell line or a plant comprising the expression vector of claim 7.

10. A method for cultivating insect-resistant and glyphosate-resistant transgenic plants, characterized in that: The steps include: (1) introducing the expression vector of claim 7 into a target plant to obtain a transgenic plant that simultaneously expresses an insect-resistant gene and a glyphosate-resistant herbicide gene; (2) screening transgenic plants with enhanced insect resistance and glyphosate resistance from the transgenic plants obtained in step (1); wherein the plant is a dicotyledonous plant or a monocotyledonous plant; The monocotyledonous and dicotyledonous plants include cotton, corn, rice, tobacco, wheat, rapeseed or alfalfa.

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