Herbicide resistance gene expression vector and application thereof

A technology for expressing vectors and resistance genes, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problems of uncertain traits, uncertain safety evaluation of expressed proteins, and low expression levels

Active Publication Date: 2021-04-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The breeding workload of the hybrid strategy is heavy, and the traits of the offspring of the two traits are uncertain; while the gene fusion and 2A peptide expression strategies have the disadvantages of low expression, uncertainty of the expressed protein, and difficulty in safety evaluation

Method used

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  • Herbicide resistance gene expression vector and application thereof
  • Herbicide resistance gene expression vector and application thereof
  • Herbicide resistance gene expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the construction of carrier

[0040] In order to construct the three vectors involved in the present invention, the p35S-OsAct1 intron composite promoter (shown in 4069bp-5393bp in SEQ ID NO: 1), the glufosinate-ammonium resistance gene bar (shown in 5401bp in SEQ ID NO: 1) were artificially synthesized respectively. -5955bp), pUBi-CP4-ter (274bp-4073bp in SEQ ID NO: 1), 35SE-pOsAct1 promoter (SEQ ID NO: 2) and the promoter pOsAct1 of rice Act1 gene (218bp in SEQ ID NO: 2 -1586bp).

[0041] (1) Construction of CSAB carrier: figure 1 , in order to obtain high expression cp4 and bar gene expression vector simultaneously, carry out double enzyme digestion to vector pCambia1300 (purchased from VWR company) with XhoI and KpnI, reclaim and obtain vector pCambia1300; NO: the plasmid of 5401bp-5955bp) in 1, obtain the bar fragment; use NcoI and KpnI to the plasmid containing the artificially synthesized p35S-OsAct1 intron composite promoter (shown in 4069bp-5393...

Embodiment 2

[0045] Embodiment 2, transformation of rice

[0046] The method of obtaining transgenic rice is to adopt the existing technology (Lu Xiongbin, Gong Zuxun (1998) Life Science 10: 125-131; Liu Fan et al. (2003) Molecular Plant Breeding 1: 108-115). The mature and plump seeds of "Xiushui-134" were dehulled, and callus was induced as transformation materials. The Agrobacterium plates of the vectors CSAB, CEAB and CAB constructed in Example 1 were taken respectively. Pick a single colony to inoculate and prepare Agrobacterium for transformation. The callus tissue to be transformed is put into OD and be 0.6 Agrobacterium bacterium liquid (preparation of Agrobacterium bacterium liquid: inoculate Agrobacterium to culture medium, be cultivated to OD be 0.6; Medium composition: 3g / L K 2 HPO 4 , 1g / LNaH 2 PO 4 , 1g / LNH 4 Cl, 0.3g / L MgSO 4 ·7H 2 O, 0.15g / L KCl, 0.01g / L CaCl 2 , 0.0025g / LFeSO 4 ·7H 20, 5g / L sucrose, 20mg / L acetosyringone, the solvent is water, pH=5.8), allow Agr...

Embodiment 3

[0047] Examination of bar and cp4 gene expression levels in embodiment 3, transgenic rice

[0048] 1. PAT / bar protein extraction and ELISA analysis

[0049] The method of checking the bar gene expression in transgenic plants: take 0.1g leaves, cut into pieces, add 1-1.5ml 1×PBS, add to the homogenizer and grind, then strictly follow the PAT / bar transgene detection kit (AS013LSEnviroLogix ) instructions can be operated.

[0050] Specifically, the sample extract was diluted 500 times with 1×PBS. The same tissue extracts from non-transgenic rice plants containing the same dilution multiples were used as a control to detect the background signal produced by the test tissue. BAR standards containing known concentrations produced by microorganisms (Li Congcong et al. 2015. Hubei Agricultural Science, 000 (010), 2516-2518) were used to generate a standard curve (the curve equation is Y=0.228X-1.638). Each plate contains a blank buffer to detect background signal from the extractio...

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Abstract

The invention discloses a herbicide resistance gene expression vector and application thereof. The expression vector comprises a promoter, a glyphosate resistance gene, a glufosinate-ammonium resistance gene and a terminator, wherein the glyphosate resistance gene adopts a promoter pUbi, the glufosinate-ammonium resistance gene adopts a composite promoter, and the composite promoter is composed of a corn Ubi promoter pUbi, a 35S promoter of cauliflower virus and an intron in a rice Actin gene. According to the invention, the glyphosate resistance gene and the glufosinate-ammonium resistance gene are highly expressed by using the same vector, and a transgenic plant with high resistance to glyphosate and glufosinate-ammonium at the same time can be obtained through the vector.

Description

[0001] (1) Technical field [0002] The invention relates to a herbicide resistance gene expression vector and its application. [0003] (2) Background technology [0004] The traits of transgenic plants are determined by the genes themselves and the regulatory elements that control gene expression. Gene expression regulatory elements include promoters, terminators, enhancers, and signal peptides, and any expression regulatory element is crucial to gene expression. Scientists in my country screened out a large number of promoters from petunia MADS-box gene flower binding protein 7 (FBP7) 2 promoter to overexpress IAA in the epidermis of cotton ovules, which increased the lint yield of transgenic cotton compared with the control Above 15%, the fiber fineness is also significantly improved, while other promoters have no such effect (ZhangM et al. 2011. Nature Biotechnology. 29(5), 453-458.). [0005] Weeds are produced along with human production activities, and their existence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29C12N15/66A01H5/00A01H6/46A01H6/20A01H6/54
CPCC12N15/8275C12N15/8277C12N15/66
Inventor 张先文梅磊王东芳项雅琴胡卫珍沈志成
Owner ZHEJIANG UNIV
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