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Artificially synthesized glyphosate-resistant mutant gene and expression vector and application thereof

A technology of expression cassettes and recombinant vectors, applied in the fields of plant molecular biology and genetic engineering, can solve problems such as differences in efficiency, and achieve the effects of strengthening water retention, important economic value, and improving glyphosate resistance

Pending Publication Date: 2019-05-17
CHONGQING ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Signal peptides from different sources have different efficiencies for EPSPS proteins to function in different transformed receptors

Method used

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  • Artificially synthesized glyphosate-resistant mutant gene and expression vector and application thereof
  • Artificially synthesized glyphosate-resistant mutant gene and expression vector and application thereof
  • Artificially synthesized glyphosate-resistant mutant gene and expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the modification synthesis of CA-MG1-EPSPS gene

[0046] 1. Source and modification of EPSPS gene

[0047] According to the characteristics of the maize genome and codon preference, the codon and coding frame of G1174-A (SEQ ID NO: 1) were modified, and the nucleotide sequence of the modified mutant gene (SEQ ID NO: 3) had a homology of 95.45 with the original EPSPS %, while the content of G changed from 27.9% to 29.2%; the content of C also changed from 41.7% to 44.8%; the content of T changed from 16.5% to 13.0%, and the content of A changed from 13.9% to % to 13.0%.

[0048] 2. Fusion protein CA-MG1-EPSPS and its coding gene

[0049] The chloroplast transit peptide CTP2 from Arabidopsis thaliana and the coded chloroplast signal peptide AHAS are added to the 5' end of the mutant gene, named fusion gene CA-MG1-EPSPS, the nucleotide sequence of the gene is sequence 4, wherein sequence 4 Positions 1-228 are the gene encoding the chloroplast transit pepti...

Embodiment 2

[0050] Example 2, Functional Verification of CA-MG1-EPSPS Gene

[0051] 1. Transgenic tobacco with CA-MG1-EPSPS gene

[0052] 1, the construction of plant expression vector (sequence 4 is the nucleotide sequence of fusion gene CA-MG1-EPSPS, and sequence 5 is to comprise the 1st-1415th rice actin 1 promoter, transcription start site, intron 1P-ract1 / ract1 intron, 1416-3161 CA-MG1-EPSPS gene and 3162-3435 Nos terminator)

[0053] The recombinant vector pTF101.1-CA-MG1-EPSPS-Bar is a eukaryotic recombinant expression vector obtained by inserting the fragment shown in Sequence 5 of the sequence listing into the SmaI restriction site of the plant expression vector pTF101.1 ( figure 1 ).

[0054] 2. Preparation of recombinant Agrobacterium

[0055] The above-mentioned recombinant vector pTF101.1-CA-MG1-EPSPS-Bar was freeze-thawed (references: Holsters M, de Waele D, Depicker A.Transfection and transformation of Agrobacterium tumefaciens.Mol Gen Genet, 1978,183:181 -187) into Agr...

Embodiment 4

[0079] Example 4, Functional Verification of CA-MG1-EPSPS Gene

[0080] 1. Transgenic CA-MG1-EPSPS maize

[0081] 1. Construction of plant expression vectors

[0082] The recombinant vector pTF101.1-CA-MG1-EPSPS-Bar is a eukaryotic recombinant expression vector obtained by inserting the fragment shown in Sequence 5 of the sequence listing into the SmaI restriction site of the plant expression vector pTF101.1.

[0083] 2. Preparation of recombinant Agrobacterium

[0084] The above-mentioned recombinant vector pTF101.1-CA-MG1-EPSPS-Bar was freeze-thawed (references: Holsters M, de Waele D, Depicker A.Transfection and transformation of Agrobacterium tumefaciens.Mol Gen Genet, 1978,183:181 -187) into Agrobacterium EHA101 to obtain recombinant Agrobacterium.

[0085] Use the primer pair consisting of primer F (5'-CGGCGACATCAAGTCCCACG-3') and primer R (5'-GGCGAAGAACTGCGGGTAGGA-3') to identify the recombinant Agrobacterium by PCR. If the target band size is 688bp, it is positive rec...

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Abstract

The invention discloses an artificially synthesized glyphosate-resistant mutant gene and an expression vector and application thereof. The invention provides a DNA fragment which is a nucleic acid molecule shown as any one of the following 1-7: 1, its coding sequence comprises a sequence 3 in a sequence table; 2, its coding sequence comprises a sequence 4 in the sequence table; 3, its coding sequence comprises the positions 427-1746 of the sequence 4 in the sequence table; 4, its coding sequence comprises a sequence 5 in a sequence table; 5, its coding sequence comprises the position 1416-3161of the sequence 5 in the sequence table; 6, it is hybridized with any defined DNA molecule in 1-5 under stringent conditions and encodes the DNA molecules of the same proteins; 7, it has the homologyof 80% or above or 90% or above with any defined DNA molecule in 1-5 and encodes the DNA molecules of the same proteins. After CA-MG1-EPSPS is introduced into tobaccos and maize, stable genetic CA-MG1-EPSPS transformants can be obtained, and the glyphosate resistance in plants can be improved.

Description

technical field [0001] The invention relates to the fields of plant molecular biology and genetic engineering, in particular to an artificially synthesized glyphosate-resistant mutant gene, its expression vector and its application. Background technique [0002] Weeds are a major problem in cultivated land, and the widespread use of herbicides has made weed control easier. Glyphosate (glyphosate) is a non-selective, organic phosphorus stem and leaf treatment herbicide developed and produced by Monsanto in 1971. It has the advantages of stable physical and chemical properties, high efficiency, low toxicity, low residue, easy to be decomposed by microorganisms, and does not damage the soil environment. It has been widely used by farmers all over the world before crops are planted. However, as a non-selective herbicide, glyphosate also has a killing effect on crops, which limits the scope and time of its use. To overcome this problem, cultivate crops that are tolerant to the b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H5/00A01H6/82
Inventor 杨小艳谢树章李新海雷开荣翁建峰王忠伟韩垚吴红
Owner CHONGQING ACAD OF AGRI SCI