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EPSP synthase gene rice chloroplast expression vector and application thereof

A chloroplast expression and chloroplast technology, applied in the application, the use of vectors to introduce foreign genetic material, the introduction of foreign genetic material and modified cells, etc. problem, to achieve the effect of good glyphosate resistance, improved glyphosate resistance, and high biosafety

Inactive Publication Date: 2012-07-11
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some EPSPS genes resistant to herbicides have been cloned from microorganisms, plants, etc., and corresponding transgenic plants have been obtained, but few of them can reach the level of promotion
So far, no new glyphosate-resistant varieties have been widely promoted in China

Method used

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  • EPSP synthase gene rice chloroplast expression vector and application thereof
  • EPSP synthase gene rice chloroplast expression vector and application thereof
  • EPSP synthase gene rice chloroplast expression vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1, acquisition of EPSP synthase gene, construction and transformation of expression vector, identification of transgenic rice

[0036] 1 Extraction of rice chloroplast genome:

[0037] (1) Remove the veins from 5-10g of rice leaves, cut the leaves into pieces, and grind them with liquid nitrogen;

[0038] (2) Add 5 times the volume of buffer A (buffer A: 50mM Tris-HCl, 20mM EDTA, 0.5M sucrose, 0.25M Vc, pH3.5) pre-cold at 4°C, homogenate and filter the filtrate with 8 layers of medical gauze to the pre- In a cold 50mL centrifuge tube, discard the residue.

[0039] (3) Centrifuge the filtrate at 1500g / min for 15min, discard the supernatant and keep the precipitate.

[0040] (4) Add 3mL bufferA to the precipitate, suspend it sufficiently, and transfer it to a 10mL centrifuge tube.

[0041] (5) Centrifuge at 2000g / min for 15min, discard the supernatant, and collect the precipitate.

[0042] (6) Add 2 mL of buffer B and suspend fully (buffer B: 50 mM Tris-HCl...

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Abstract

The invention discloses an EPSP synthase (5-enolpyruvyl-shikimate-3-phosphate synthase) gene rice chloroplast expression vector and application of the expression vector. The rice chloroplast expression vector comprises EPSP synthase gene expression cassette, and homologous recombination fragments atpB and rbcL in rice chloroplast genome, wherein the EPSP synthase gene expression cassette is located between atpB and rbcL. The invention also provides a construction method of the chloroplast expression vector. According to the invention, the rice chloroplast expression vector is applied to improve glyphosate resistance of rice, and the application comprises transforming rice callus or callus cells with rice chloroplast expression vector and regenerating to obtain genetically modified rice with notably improved glyphosate resistance. The rice chloroplast expression vector of the invention can efficiently improve glyphosate resistance in the genetically modified rice to obtain genetically modified rice with stable heredity and without gene silencing or gene segregation, and has the advantages of high safety, time saving effect, high efficiency, low energy consumption, low cost and the like.

Description

technical field [0001] The present invention relates to a chloroplast expression vector, in particular to an EPSP synthase (5-enol-pyruvylshikimate-3-phosphate synthase) gene rice chloroplast expression vector and a construction method thereof, and the present invention further relates to the EPSP synthase The application of gene rice chloroplast expression carrier in improving rice glyphosate resistance belongs to the field of transgenic rice. Background technique [0002] In 1988, Boynton et al. bombarded atpB-mutant Chlamydomonas with chloroplast DNA carrying atpB wild-type gene for the first time by gene gun method, which restored the ability of photosynthesis, thus marking the beginning of chloroplast genetic engineering. In 1900, the rrn16 gene was expressed in tobacco chloroplasts for the first time by using the gene gun method, which opened the curtain of chloroplast transformation in higher plants. Grass plants such as rice, corn and wheat are important food crops,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10C12N15/66A01H5/00
Inventor 李轶女张志芳王金辉王国增程奇倪丕冲沈桂芳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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