Corn insect-resistant and herbicide-resistant gene and application thereof in cultivation of insect-resistant and herbicide-resistant corn

Abstract

The invention provides an insect-resistant and herbicide-resistant gene of corn and application of the insect-resistant and herbicide-resistant gene to cultivation of insect-resistant and herbicide-resistant corn, and belongs to the field of plant disease resistance. Experiments prove that the expression vector for simultaneously expressing the Cry1Ab, Cry2Ab and cp4-epsps genes is constructed for the first time, transgenic crops obtained by transforming crops with the vector can simultaneously resist Lepidoptera pests such as ostrinia nubilalis and cotton bollworm and have good glyphosate resistance, the insect-resistant and herbicide-resistant application of the transgenic crops is widened, and the transgenic crops have broad application prospects. The method has a wide application prospect in corn genetic breeding improvement.

Description

Technical Field

[0001] This invention relates to the field of plant technology, and more specifically, to a gene for insect-resistant and herbicide-tolerant maize and its application in breeding insect-resistant and herbicide-tolerant maize. Background Technology

[0002] Genetically modified (GM) corn is a type of genetically modified crop. Genetic engineering utilizes modern biotechnology to artificially clone genes representing desired traits and then transfer them into recipient organisms through transgenic manipulation, thereby improving the recipient organism's original traits or endowing it with new, desirable traits. GM corn, as a significant achievement in the industrialization of biotechnology, is widely used around the world.

[0003] Lepidoptera pests are highly damaging insects. Common lepidopteran species include rice leaf roller, beet armyworm, cotton bollworm, peach fruit moth, bollworm, diamondback moth, melon leafminer, and bean pod borer. Once severe resistance develops, it can cause significant losses, confusion, and increased control costs for farmers.

[0004] Glyphosate is a chemical substance, a non-selective, residue-free post-emergence herbicide. It is highly effective against deep-rooted perennial weeds and annual and biennial grasses, sedges, and broadleaf weeds. Glyphosate is usually formulated as isopropylamine salt or sodium salt for use. Its weeding effect is excellent, primarily absorbed through the leaves, with a small amount absorbed through the roots and transported to the plant's growing point. Glyphosate inhibits the enzymes required for the synthesis of three aromatic amino acids (tyrosine, tryptophan, and phenylalanine), thus it is primarily effective against plants in their active growth phase and is not suitable as a pre-emergence herbicide. This herbicide is highly effective against both annual and perennial weeds. Through genetic modification, glyphosate-tolerant crops can be bred.

[0005] While there have been reports on technologies for insect-resistant and herbicide-tolerant maize, research on their effectiveness in preventing lepidopteran pests and improving glyphosate tolerance is limited. There is an urgent need to provide a transgenic maize variety that effectively combines both of these properties. Summary of the Invention

[0006] To address the technical problems existing in the prior art, this invention provides a gene for insect-resistant and herbicide-tolerant maize and its application in breeding insect-resistant and herbicide-tolerant maize. Experiments have confirmed that this invention, for the first time, constructs an expression vector simultaneously expressing the Cry1Ab, Cry2Ab, and cp4-epsps genes. Transgenic crops obtained by transforming crops with this vector exhibit resistance to lepidopteran pests such as the corn borer and cotton bollworm, and also possess good resistance to glyphosate. This broadens the application of insect-resistant and herbicide-tolerant transgenic crops and has wide-ranging application prospects for maize genetic breeding improvement.

[0007] Specifically, one of the objectives of this invention is to provide a fusion gene for corn that is resistant to insects and herbicides, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Another objective of the present invention is to provide a fusion gene expression vector comprising the fusion gene as shown in SEQ ID NO: 1.

[0009] Another objective of this invention is to provide a method for constructing the above-mentioned fusion gene expression vector, comprising the following steps: 1) PCR amplification of the fusion gene yields the amplified fusion gene fragment; 2) After digesting the pBI221 plasmid and the fusion gene amplification fragment prepared in step 1), they were ligated to obtain the ligation product; 3) Transform the ligation product into E. coli DH5α, select positive clone vectors, extract plasmids, and name the plasmid with correct enzyme digestion and sequencing results as pBI221-Cry1Ab-Cry2Ab-cp4-epsps, which is the fusion gene expression vector.

[0010] Preferably, the primers used for PCR amplification in step 1) are: P1: 5'-GCTCTAGAATGGATAACAATCCGAACA-3' (SEQ ID NO: 2); P2: 5'-GCGGATCCGCTGTAGCCACTGATGCTG-3' (SEQ ID NO: 3) The underlined areas are enzyme cleavage sites.

[0011] Preferably, the PCR amplification reaction system in step 1) includes: 2.5 μL 10× buffer, 2 μL 25 mmol / L MgCl2, 2 μL 10 mmol / L dNTP, 1.0 U Taq DNA polymerase, 1 μL each of 10 μM primers, and an appropriate amount of double-distilled water to 25 μL.

[0012] Preferably, the PCR amplification reaction conditions in step 1) are 94℃ / 5min; 94℃ / 30s, 58℃ / 1min, 72℃ / 45s, 35 cycles; 72℃ / 10min.

[0013] Preferably, the enzyme digestion in step 2) is XbaI / BamHI double digestion.

[0014] Preferably, the enzyme digestion reaction system in step 2) includes: 10 μL plasmid DNA (or fusion gene), 10 μL 10× buffer, 5 μL XbaI, 5 μL BamHI, and an appropriate amount of double-distilled water to 50 μL; digest overnight at 30°C, and take 5 μL of sample for electrophoresis to check whether the enzyme digestion is complete. The completely digested sample is then recovered by column chromatography.

[0015] Preferably, the ligation system in step 2) is: 2.0 μL of pBI221 plasmid digested with enzymes, 4.0 μL of fusion gene digested with enzymes, 5.0 μL of 2× buffer, and 2 μL of T4 DNA ligase; the ligation conditions are: ligation reaction at 16℃ for 10 h.

[0016] Preferably, the enzymatic digestion reaction in step 3) is the same as in step 2).

[0017] Another objective of this invention is to provide a fusion gene engineered bacterium, which is prepared by transferring a fusion gene expression vector into a host; the fusion gene expression vector contains a nucleotide sequence as shown in SEQ ID NO: 1.

[0018] Preferably, the transformation method can be Agrobacterium-mediated transformation, gene gun method, protoplast infection method or other plant genetic transformation methods, with Agrobacterium-mediated transformation being the preferred method.

[0019] The host selected is Agrobacterium LBA4404, EHA105, GV3101, AGL-1 or EHA101.

[0020] The fusion gene expression vector constructed in this invention is suitable for expression in monocotyledonous plants or dicotyledonous plants. Monocotyledonous plants include corn, rice, etc.; dicotyledonous plants include soybean, rapeseed, cotton, etc.

[0021] The plants containing the fusion gene are resistant to pests, including lepidopteran pests such as corn borers and cotton bollworms.

[0022] The plant containing the fusion gene exhibits glyphosate tolerance.

[0023] Beneficial effects: This invention provides a gene for insect-resistant and herbicide-tolerant maize and its application in breeding insect-resistant and herbicide-tolerant maize. This invention is the first to construct an expression vector that simultaneously expresses the Cry1Ab, Cry2Ab, and cp4-epsps genes. The transgenic crops obtained by transforming crops with this vector can simultaneously resist lepidopteran pests such as corn borer and cotton bollworm and have good resistance to glyphosate. This expands the application of insect-resistant and herbicide-tolerant transgenic crops and has broad application prospects for maize genetic breeding improvement. Attached Figure Description

[0024] Figure 1This is the identification of T1 generation transgenic maize plants. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0026] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0027] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0028] Example 1 A fusion gene for insect resistance and herbicide tolerance in maize, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0029] A fusion gene expression vector containing the fusion gene as shown in SEQ ID NO: 1.

[0030] The method for constructing the above-mentioned fusion gene expression vector includes the following steps: 1) PCR amplification of the fusion gene yields the amplified fusion gene fragment; 2) After digesting the pBI221 plasmid and the fusion gene amplification fragment prepared in step 1), they were ligated to obtain the ligation product; 3) Transform the ligation product into E. coli DH5α, select positive clone vectors, extract plasmids, and name the plasmid with correct enzyme digestion and sequencing results as pBI221-Cry1Ab-Cry2Ab-cp4-epsps, which is the fusion gene expression vector.

[0031] In step 2), the enzyme digestion is performed using XbaI / BamHI double digestion.

[0032] The primers used for PCR amplification in step 1) are: P1: 5'-GCTCTAGAATGGATAACAATCCGAACA-3' (SEQ ID NO: 2); P2: 5'-GCGGATCCGCTGTAGCCACTGATGCTG-3' (SEQ ID NO: 3) The underlined areas are enzyme cleavage sites.

[0033] The PCR amplification reaction system in step 1) includes: 2.5 μL 10× buffer, 2 μL 25 mmol / L MgCl2, 2 μL 10 mmol / L dNTP, 1.0 U Taq DNA polymerase, 1 μL each of 10 μM primers, and an appropriate amount of double-distilled water to 25 μL. The PCR amplification reaction conditions in step 1) are 94℃ for 5 min; 94℃ for 30 s, 58℃ for 1 min, 72℃ for 45 s, 35 cycles; 72℃ for 10 min.

[0034] The enzyme digestion reaction system in step 2) includes: 10 μL plasmid DNA (or fusion gene), 10 μL 10× buffer, 5 μL XbaI, 5 μL BamHI, and an appropriate amount of double-distilled water to 50 μL; digest overnight at 30℃, and take 5 μL of sample for electrophoresis to check whether the enzyme digestion is complete. The completely digested sample is then recovered by column chromatography.

[0035] The ligation system in step 2) was as follows: 2.0 μL of pBI221 plasmid digested with enzymes, 4.0 μL of fusion gene digested with enzymes, 5.0 μL of 2× buffer, and 2 μL of T4 DNA ligase; the ligation conditions were: ligation reaction at 16℃ for 10 h.

[0036] The enzymatic digestion reaction described in step 3) is the same as in step 2).

[0037] A fusion gene engineered bacterium, prepared by transferring a fusion gene expression vector into a host; the fusion gene expression vector contains a nucleotide sequence as shown in SEQ ID NO: 1.

[0038] The transformation method is selected from Agrobacterium-mediated transformation, gene gun method, protoplast infection method or other plant genetic transformation methods.

[0039] The host selected is Agrobacterium LBA4404, EHA105, GV3101, AGL-1 or EHA101.

[0040] The fusion gene expression vector constructed in this invention is suitable for expression in monocotyledonous plants or dicotyledonous plants. Monocotyledonous plants include corn, rice, etc.; dicotyledonous plants include soybean, rapeseed, cotton, etc.

[0041] The plants containing the fusion gene are resistant to pests, including lepidopteran pests such as corn borers and cotton bollworms.

[0042] The plant containing the fusion gene exhibits glyphosate tolerance.

[0043] Example 2 2.1 Obtaining Transgenic Insect-Resistant and Herbicide-Tolerant Maize Following the procedures described in the known references (Ishida Y, Saito H, Ohta S, et al. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol, 1996, 14: 745-750. Sidorov V, Duncan D. Agrobacterium-mediated maize transformation: immature embryos versus callus. Methods Mol Biol, 2009, 526: 47-58.), the fusion gene vector prepared in Example 1 was used to infect immature maize embryos (1.0-1.5 mm in size) using Agrobacterium-mediated transformation, and transgenic maize plants were obtained after induction and differentiation screening.

[0044] Identification of T1 generation transgenic maize plants: qPCR was used to detect whether the T1 generation of the above transgenic maize plants contained the fusion gene. The specific steps are as follows. Genomic DNA was extracted from leaves of T1 generation transgenic maize and non-transgenic maize using the CTAB method and used as templates for PCR reactions. Upstream and downstream primers were designed based on the Cry1Ab, Cry2Ab, and cp4-epsps gene fragments. Cry1Ab-F:5'-TACAATTACGTTTGTATACTA-3'(SEQ ID NO.4), Cry1Ab-R:5'-GTATCCTACAGGAGAAGCCAT-3'(SEQ ID NO.5) Cry2Ab-F:5'-TACGTTTTGTATAATGAAATAAGAAATA-3'(SEQ ID NO.6), Cry2Ab-R:5'-TGTCATTTGGCGCTAAATGA-3'(SEQ ID NO.7) cp4-epsps-F:5'-CTACAATTACGTTTGTCAAAGATGG-3'(SEQ ID NO.8), cp4-epsps-R:5'-CTTGCGAAGGATAGTGGGAT-3'(SEQ ID NO.9) Among them, the upstream primers 5' of Cry1Ab, Cry2Ab, and cp4-epsps were labeled with FAM, HEX, and TET luminescent groups, respectively.

[0045] The PCR reaction system is as follows: 10×PCR Buffer 10μL, dNTPs 5μL, upstream primers of each gene 3μL, downstream primers of each gene 3μL, maize genomic DNA 2μL, Taq DNA polymerase 1μL, and ddH2O to a final volume of 50μL.

[0046] Reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; final extension at 72℃ for 10 min.

[0047] See results Figure 1 The transgenic maize plants showed obvious fluorescent signals, while the non-transgenic maize plants did not, thus confirming that the transgenic maize plants described in this invention can normally express the fusion gene and can effectively integrate into the maize chromosome.

[0048] 2.2 Evaluation of Insect Resistance Performance of Transgenic Insect-Resistant and Herbicide-Tolerant Maize After 25 days of germination at 37℃, 100 plants from different generations of transgenic and non-transgenic maize seeds were selected for cultivation, with 20 plants per group. Ten plants were inoculated with 10 fourth-instar corn borers each, and the other 10 plants were inoculated with 10 fourth-instar cotton bollworms each. Mortality was observed after 7 days. The test results are shown in Table 1.

[0049] Table 1 Evaluation of insect resistance performance of transgenic insect-resistant and herbicide-tolerant maize As shown in Table 1, the genetically modified corn of the present invention has excellent insect resistance and can effectively kill lepidopteran pests such as corn borers and cotton bollworms.

[0050] 2.3 Evaluation of herbicide resistance performance of transgenic insect-resistant and herbicide-tolerant maize T2 generation transgenic maize and non-transgenic maize seeds were sprayed with 15% glyphosate (Shandong Suitai Biotechnology Co., Ltd.) diluted with tap water at volume ratios of 1:40, 1:80, and 1:240, respectively, at a dosage of 50 L / mu, 25 days after germination and at the 6-leaf stage. The growth and development and mortality of maize were recorded 7 days later. The results are shown in Table 2.

[0051] Table 2 Evaluation of herbicide resistance performance of transgenic insect-resistant and herbicide-tolerant maize As shown in Table 2, the genetically modified corn of the present invention has excellent herbicide resistance, can effectively resist glyphosate, and has high tolerance.

[0052] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A corn insect-resistant herbicide-tolerant fusion gene, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

1.

2. A fusion gene expression vector, characterized by, The fusion gene shown as SEQ ID NO: 1 is contained.

3. The method for constructing the fusion gene expression vector according to claim 2, wherein, The method comprises the following steps: 1) PCR amplification of the fusion gene to obtain a fusion gene amplification fragment; 2) after pBI221 plasmid and the fusion gene amplification fragment prepared in step 1) are cut, linking is performed to obtain a linking product; 3) the linking product is transformed into E. coli DH5a, positive cloning vectors are selected, plasmids are extracted, and the plasmid with correct enzyme cutting and sequencing results is named as pBI221-Cry1Ab-Cry2Ab-cp4-epsps, which is the fusion gene expression vector.

4. A fusion genetically engineered bacterium, characterized by, The genetically engineered bacteria are prepared by transforming a host with a fusion gene expression vector; the fusion gene expression vector contains the nucleotide sequence shown as SEQ ID NO:

1.

5. The genetically engineered bacteria as described in claim 4, characterized in that, The method for transformation can be Agrobacterium-mediated transformation, biolistic transformation, protoplast infection or other plant genetic transformation methods.

6. The genetically engineered bacteria as described in claim 4, characterized in that, The host is selected from Agrobacterium LBA4404, EHA105, GV3101, AGL-1 or EHA101.

7. Use of the corn insect-resistant and herbicide-resistant fusion gene of claim 1 in the preparation of a transgenic plant.

8. Use according to claim 7, wherein the compound is ###0002### The plant includes corn, rice, soybean, rape or cotton.

9. The use according to claim 7, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The transgenic plant has pest resistance, including corn borer, cotton bollworm and other lepidopteran pests.

10. The use according to claim 7, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The transgenic plant has glyphosate tolerance.

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