Pest-resistant gene T-DNA vector and application thereof

A technology of insect-resistant genes and vectors, applied in recombinant DNA technology, application, genetic engineering, etc., can solve the problem of narrow insecticidal spectrum and achieve the effect of increased expression

Inactive Publication Date: 2018-12-14
ZHEJIANG UNIV
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  • Application Information

AI Technical Summary

Problems solved by technology

A single insecticidal toxin often has a relatively narrow insecticidal spectrum; at the same time, long-term use o

Method used

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  • Pest-resistant gene T-DNA vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, construction of insect-resistant gene expression cassette

[0022] The nucleotide sequence of the anti-insect gene Cry1Ab artificially synthesized by Shanghai Sangong is SEQ ID NO:1, and the nucleotide sequence of Vip3A is SEQ ID NO:2. gene, the sequence of the insect resistance gene is SEQ ID NO:3. The nucleotide sequence of the promoter CaMV 35S artificially synthesized by Shanghai Sangong is SEQ ID NO: 4, and the nucleotide sequence of the intron Actin-intron encoding rice actin is SEQ ID NO: 5.

[0023] The construction process of the insect-resistant gene: the Cry1Ab fragment was obtained by digestion with BamHI and XmaI, and the Vip3A fragment was obtained by digestion with XmaI and SacI. The above two fragments were ligated into the intermediate vector with T4 DNA polymerase to obtain a complete insect-resistant gene with restriction sites at both ends being BamHI and SacI.

[0024] The construction process of the promoter 35S / Actin-intron: the Ca...

Embodiment 2

[0026] Embodiment 2, the acquisition of transgenic corn

[0027] The method used in the maize transformation event is the Agrobacterium-mediated method, and the transformation is carried out according to the method and medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), and the specific steps are as follows: take 8-10 days after pollination Hi-2 ear of corn. All immature embryos (1.0-1.5 mm in size) were collected, and Agrobacterium containing the T-DNA vector pCam-1Ab-v3A constructed in Example 1 was co-cultured with the immature embryos for 2-3 days (22° C.). Transfer the immature embryos to the callus induction medium (containing 200 mg / L timentin Agrobacterium bacterium), and culture in the dark at 28°C for 10-14 days. All calli were transferred to selection medium with 2.0 mM glyphosate, and cultured in the dark at 28°C for 2-3 weeks.

[0028] Transfer all tissues to fresh selection medium and culture in dark at 28°C for 2-3 weeks. Then transfer ...

Embodiment 3

[0030] Embodiment 3, the mensuration of insect resistance ability of transgenic crops

[0031] The insect resistance activity of the 10 transgenic maize lines obtained in Example 2 was measured by corn borer and cotton bollworm. The young leaves of the transgenic corn obtained in Example 2 were fed to first-instar larvae, inoculated with 10 larvae per leaf, and cultured in the dark at 28°C for 5 days to record the data. After the first instar larvae of newborn corn borer fed on 10 transgenic corn lines, all of them died 100%; after the first instar larvae of newborn cotton bollworm fed on 10 transgenic corn lines, 8 lines of cotton bollworms died 100%, and the other two lines The mortality rate of cotton bollworm is between 60% and 90%.

[0032] At the same time, by the method of Example 2, the T-DNA of the control group CK was transferred into the corn to obtain the control transgenic corn line in which pOsActin1 promotes the expression of the insect resistance gene. The an...

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Abstract

The invention discloses construction of a pest-resistant gene T-DNA vector and application thereof. A compound promoter is used for promoting expression of a pest-resistant gene; the compound promotercontains a cauliflower mosaic virus (CaMV) 35S promoter and an intron sequence of rice actin Actin1; the pest-resistant is formed by connecting Cry1Ab and Vip3A through a connecting peptide. After being built, a recombinant vector is transferred into corn, so that the expression volume of pest-resistant protein in transgenic maize is obviously increased. The recombinant vector disclosed by the invention can be used for preparing cells of a pest-resistant plant; the pest-resistant plant comprises maize, rice and the like. A transgenic plant transformed from the recombinant vector disclosed bythe invention can efficiently kill main lepidoptera pests such as armyworm, prodenia litura, european corn borer, cotton bollworm and beet armyworm.

Description

(1) Technical field [0001] The invention relates to the construction and application of an insect-resistant gene T-DNA carrier. (2) Background technology [0002] Pests cause an annual loss of about 8 billion US dollars to global agricultural production. The control of pests currently mainly relies on the use of chemical pesticides, but the residues of pesticides will have adverse effects on human health and the environment. The use of biotechnology to cultivate transgenic insect-resistant crops containing insect-resistant genes can greatly reduce the use of chemical pesticides and effectively protect crops from pests. At present, transgenic insect-resistant corn and cotton have been widely planted. [0003] The key technology for transgenic crops to resist insects is to obtain insecticidal proteins with excellent performance, and there are many kinds of insecticidal proteins. More commonly used are BT insecticidal crystal proteins, such as Cry1Ab, Cry1C, etc., which have b...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/46
CPCC12N15/8251C12N15/8286
Inventor 于小星许超沈志成
Owner ZHEJIANG UNIV
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