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Plant expression vector containing glyphosate resistant gene and application thereof

A glyphosate-resistant and expression vector technology, applied in the field of genetic engineering, can solve problems such as phytotoxicity, non-resistance to herbicides, and damage to cotton plants.

Active Publication Date: 2017-01-11
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently promoted cotton varieties are not resistant to herbicides, and phytotoxicity often occurs, causing certain damage to cotton plants. Breeding herbicide-resistant crop varieties is the most effective way to prevent herbicide phytotoxicity

Method used

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  • Plant expression vector containing glyphosate resistant gene and application thereof
  • Plant expression vector containing glyphosate resistant gene and application thereof
  • Plant expression vector containing glyphosate resistant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Bivalent mc2-epsps plant expression vector construction

[0029] Transformation of plant expression vector: Using pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) as a template, amplify the sequence of the kanamycin resistance gene (aadA) to the plasmid replication origin (pVS1ori), at the 5' end EcoRI and PvuI restriction sites were added, ScaI and EcoRI restriction sites were added at the 3' end, the primer sequences were SEQ ID NO: 7 and SEQ ID NO: 8, and the amplified fragment was named Fragment A. Fragment A was digested with EcoRI enzyme, and vector p3 was obtained by self-circularization. Synthesize LB to RB end sequences, in order to increase the proportion of positive plants in genetic transformation, reduce the incidence of vector backbone DNA in the genome of transgenic plants, and increase the frequency of low-copy transformation events, modify the LB and RB ends, and add recognition cutting elements inside , adding ...

Embodiment 2

[0033] The acquisition of embodiment 2 transmutation bivalent mc2-epsps gene cotton

[0034] The Agrobacterium-mediated method used here is a plant genetic transformation method well known to those skilled in the art. The specific operation procedure is:

[0035] 1. Strain culture

[0036] The constructed plant expression vector of glyphosate-resistant gene was transformed into Agrobacterium strain LBA4404 by electric shock, and a single colony of Agrobacterium was inoculated in the culture medium containing 50 mg / L kanamycin (km), rifampicin (rifampicin, rif). 25mg / L of LB (tryptone 5g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L) or YEB (beef extract 5g / L, yeast powder 1g / L, peptone 5g / L, sucrose 5g / L, MgSO 4 5ml / L) in liquid medium. Incubate overnight at 28°C in the dark with shaking to the logarithmic phase of bacterial growth. Dilute the bacterial solution with LB or YEB liquid medium, shake and culture for 4-6 hours, and dilute the bacterial solu...

Embodiment 3T0

[0049] Example 3 T0 Generation Transgenic Cotton Resistance Identification

[0050] After the grafting and transplanting of transgenic seedlings in Example 2 survived, spray 3000ppm glyphosate solution (glyphosate commercial preparation Roundup: Monsanto) in the 6-8 leaf stage, spray 45ml per square meter (i.e. 30L / mu) The symptoms were observed 7-14 days after each spraying, and the non-transgenic recipient material Jimian 14 was used as a negative control. 69 transformants of the transgenic cotton transformed with the bivalent mc2 gene showed resistance to glyphosate. Resistant plants were strictly self-pollinated. Due to the influence of tissue culture operations and greenhouse cultivation conditions, some of the resistant plants had poor fertility. 52 lines of T1 generation inbreds were received.

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Abstract

The invention discloses a plant expression vector containing glyphosate resistant gene. The plant expression vector is an annular carrier containing two copies of glyphosate resistant gene; the glyphosate resistant gene coded amino acid sequence is the protein shown as SEQ ID NO.1 or glyphosate resistant protein that is substituted and / or deleted and / or added with one or several amino acid residues compared with SEQ ID NO.1. The two copies of glyphosate resistant gene are respectively driven by a 1, 5-ribulose bisphosphate carboxylase gene promoter from cotton and a promoter from arabidopsis thaliana actin gene. The invention proves that transgenic cotton introducing the plant expression vector has very strong glyphosate resistance. The plant expression vector provided by the invention is of great significance for further cultivation of high glyphosate resistant plants and application thereof.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a plant expression vector containing a glyphosate-resistant gene. Background technique [0002] Glyphosate is a non-selective herbicide with stable physical and chemical properties, high efficiency, broad spectrum, low toxicity, low residue, easy to be decomposed by microorganisms, and does not damage the soil environment. It has been widely used in agricultural production and has become the current It is the most widely produced pesticide variety in the world. Since the successful development of the glyphosate-based herbicide Roundup by Monsanto Company in the United States in 1976 and its wide application, research on transgenic crops resistant to glyphosate has become a hot spot in the research of herbicide-resistant genetic engineering. With the development of glyphosate-resistant gene cloning, glyphosate-resistant transgenic crops have also come out and been popularized and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N5/10C12N1/21A01H5/00
Inventor 何云蔚崔洪志王建胜
Owner BIOCENTURY TRANSGENE CHINA
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