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Glyphosate-resistant gene, special expression vector and application of glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat

A glyphosate-resistant, expression-cassette technology, applied in the field of obtaining glyphosate-resistant transgenic wheat, can solve problems such as limiting the scope of use and use time

Active Publication Date: 2014-08-27
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as a non-selective herbicide, it kills weeds and also kills crops, which limits its range of use and time of use to a certain extent.

Method used

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  • Glyphosate-resistant gene, special expression vector and application of glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat
  • Glyphosate-resistant gene, special expression vector and application of glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat
  • Glyphosate-resistant gene, special expression vector and application of glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, optimization of GR79 gene and synthetic fusion gene wCTP:GR79m

[0038] The GR79 gene was isolated from a glyphosate-resistant strain in an extremely polluted environment, provided by the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The following aspects are mainly considered during optimization: wheat codon bias, GC content, and guide peptide. The guide peptide of the present invention is the chloroplast guide peptide (wCTP) from wheat RubisCO small subunit. See the gene sequence and GC content characteristics before and after optimization. The homology between the sequence optimized according to wheat codon bias and the original nucleotide sequence is 73.62%, and the codon usage before and after optimization is as follows: Figure 7 .

[0039] The fusion gene of the optimized GR79m gene and the gene combination of chloroplast guide peptide (wCTP) is named wCTP:GR79m, and its nucleotide sequence is the 2192-3675th nucleotide fr...

Embodiment 2

[0042] Embodiment 2, the application of wCTP:GR79m gene in anti-glyphosate

[0043] 1. Construction of pCS167-wCTP:GR79m overexpression vector

[0044] The specific construction method of pCG185-UMN vector is as follows: amplify about 2500bp Ubi:MCS:Nos fragment from pAHC-PSK, add PacI sites at both ends of the primers, insert the PacI sites in pCG185T-DNA after digestion with PacI , to obtain the intermediate vector pCG185-UMN.

[0045] The optimized gene was synthesized by Shanghai Sangon and cloned into the pUC57 vector to obtain the vector plasmid pUC57-wCTP:GR79. Digest pUC57-wCTP:GR79 with SpeI to recover a 1503bp wCTP:GR79m fragment, connect the recovered fragment to the pCG185-UMN vector with SpeI digestion to remove P, and obtain an expression vector driven by the Ubiqutin promoter, named pCG185-wCTP:GR79m, and construct Process such as figure 1 a.

[0046] The recombinant plasmid pCG185-wCTP:GR79m was identified by digestion with SpeI, and the results were as fol...

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Abstract

The invention discloses a glyphosate-resistant gene, a special expression vector and an application of the glyphosate-resistant gene to preparation of glyphosate-resistant transgenic wheat. The invention provides a protein which is (a) or (b): (a) a protein consists of an amino acid sequence shown in a sequence 3 in a sequence table; and (b) a protein which is derived from the amino acid sequence shown in the sequence 3 in the sequence table through the substitution and / or deficiency and / or addition of one or more amino acid residues and derived from a sequence 2 related to glyphosate resistance. Experiments prove that artificial design is carried out on wheat by utilizing the condon preference of the wheat and the chloroplast targeting sequence from wheat RubisCO small subunit is added at N segment to obtain a modified gene through an artificial synthesis method; the modified gene is applied as a selection marker gene in wheat transformation so as to obtain the glyphosate-resistant transgenic wheat; and researches prove that the resistance of the transgenic wheat is higher than the transgenic resistance of the wheat before optimization.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a glyphosate-resistant gene, a special expression vector and its application in obtaining glyphosate-resistant transgenic wheat. Background technique [0002] Glyphosate is the main active ingredient of the herbicide Roundup produced by Monsanto in the United States. It has high efficiency, broad spectrum, low toxicity, low residue, easy to be decomposed by microorganisms, does not damage the soil environment, and is destructive to most plants. Etc. Since it was successfully developed in 1976, it has been widely used in the agricultural field and has become the largest pesticide variety in the world today. Glyphosate is a systemic herbicide that can be absorbed by the plant after spraying on the stems and leaves of the plant, and quickly transported to the whole plant and its roots, thereby killing weeds. It can not only kill the above-ground parts, And it can act on the roots, tru...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/84C12N1/21C12N15/11A01H5/00
Inventor 夏兰琴王根平林敏陆伟
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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