Pest and glyphosate resistant expression vector, and plasmid and application thereof

An expression carrier and glyphosate-resistant technology, which is applied in the direction of using vectors to introduce foreign genetic material, application, and cells modified by introducing foreign genetic material, etc., to achieve the effect of delaying pest resistance and broadening the insecticidal spectrum

Active Publication Date: 2017-07-04
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Analysis of existing articles and patents found that there is no report of constructing cry1Ab, cry2Ab and G10evo genes in the same plant transformation vector

Method used

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  • Pest and glyphosate resistant expression vector, and plasmid and application thereof
  • Pest and glyphosate resistant expression vector, and plasmid and application thereof
  • Pest and glyphosate resistant expression vector, and plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, construction of expression vectors for insect-resistant protein coding genes cry1Ab, cry2Ab and glyphosate resistance gene G10evo

[0032] The plant transformation vector was constructed based on the pCambia 1300 (CAMBIA, Canberra, Australia) vector. The glyphosate resistance gene G10evo contains the nucleotide sequence of the maize acetohydroxyacid synthase chloroplast signal peptide (SEQ ID No: 3, the 5' end is designed with a BamHI restriction site, and the 3' end is designed with an Xho Ⅰ position point, BamHI-Xho Ⅰ fragment) and CaMV35S terminator (SEQ ID No: 4, Xho Ⅰ site is designed at the 5' end, HindIII site is designed at the 3' end, Xho Ⅰ-HindIII restriction fragment), A BamHI-HindIII fragment including the gene (with signal peptide) and terminator was obtained.

[0033] The 35S promoter of CaMV (containing the intron of rice actin) p35s-actinintron, using the PMD19-35Sints plasmid kept in the laboratory as a template, using the primer 35S-KpnIF (...

Embodiment 2

[0042] Example 2, the acquisition of transgenic insect-resistant and herbicide-resistant corn

[0043] 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). The specific steps are as follows: take 8-10 days after pollination Hi-2 ear of corn. Collect all the immature embryos (1.0-1.5 mm in size), and transfer the T-DNA vector pCam-pZmUbi-1-cry1Ab-PepcT-pActin-cry2Ab-T35S-p35s-intron-

[0044] G10evo-T35S Agrobacterium and immature embryos were co-cultivated for 2-3 days (22°C). Transfer the immature embryos to the callus induction medium (containing 200 mg / L timentin Agrobacterium), and culture in the dark at 28°C for 10-14 days. All the calli were transferred to the selection medium with 50ng / ml hygromycin, and cultured in the dark at 28°C for 2-3 weeks.

[0045] All the tissues were transferred to th...

Embodiment 3

[0046] Embodiment 3, the acquisition of transgenic insect-resistant and herbicide-resistant rice

[0047] The transgenic plants were obtained according to the methods and medium formulations in the literature (Lu Xiongbin, Gong Zuxun, 1998 Life Sciences 10: 125-131; Liu Fan et al., 2003 Molecular Plant Breeding 1: 108-115). The mature and plump seeds were selected to be hulled, and callus was induced to be used as transformation materials. Take the Agrobacterium plate containing the T-DNA vector pCam-pZmUbi-1-cry1Ab-PepcT-pActin-cry2Ab-T35S-p35s-intron-G10evo-T35S, pick a single colony for inoculation, and prepare the Agrobacterium for transformation. Put the callus to be transformed into the appropriate concentration of Agrobacterium solution (containing acetosyringone), let the Agrobacterium bind to the surface of the callus, then transfer the callus to the co-culture medium, and co-culture for 2-3 sky. Wash the transformed callus with sterile water, transfer to the select...

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Abstract

The invention discloses a pest and glyphosate resistant expression vector, and a plasmid and an application thereof. The expression vector contains a pest-resistant gene cry1Ab, a pest-resistant gene cry2Ab and a glyphosate-resistant gene G10evo. The vector simultaneously expresses Cry1Ab, Cry2Ab and G10evo proteins, and transgenic crops obtained by transferring crops with the vector can simultaneously resist cotton bollworms, beet armyworms, corn borers, striped rice borer and other main lepidoptera pests of rice, corn and cotton, so the insecticidal spectrum is widened, the pest resistance is effectively delayed, the transgenic crops have glyphosate resistance, the transgenic crops with composite properties accord with the current development direction of transgenic crops, and demands of large-scale agricultural production are met.

Description

[0001] (1) Technical field [0002] The invention belongs to the field of plant genetic engineering and relates to an expression vector constructed of two insect-resistant genes and a glyphosate-resistant gene. [0003] (2) Background technology [0004] Agricultural pests are the number one important factor affecting crop production. At present, chemical pesticides are still the main role in pest control and play an important role in maintaining the stable development of agricultural production. Pesticides use a non-specific poisoning method, which not only kills target pests, but also poisons beneficial insects and birds. Long-term use will cause serious pollution to the natural environment and destroy the ecological balance; due to the enrichment in the food chain, it will directly pose a threat to the safety of humans and animals. [0005] Weeds are the second most important factor affecting crop production. It competes with crops for water resources, fertilizers, light ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10A01H5/00
CPCC07K14/325C07K14/415C12N15/8205C12N15/8275C12N15/8286
Inventor 王鹏飞赵宇刘苗苗沈志成
Owner ZHEJIANG UNIV
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