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Glyphosate-resistant EPSP synthetase MC1-EPSPS, coding gene for same and application

A technology of fusion gene and gene, applied in application, genetic engineering, enzyme and other directions, can solve the problem of low expression efficiency, achieve the effect of enhancing expression, important economic value, and improving glyphosate resistance

Active Publication Date: 2018-07-27
CHONGQING ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that due to the large difference between bacterial codon bias and plant codon bias, if the EPSPS gene from bacteria is directly transformed into plants, its expression efficiency is low

Method used

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  • Glyphosate-resistant EPSP synthetase MC1-EPSPS, coding gene for same and application
  • Glyphosate-resistant EPSP synthetase MC1-EPSPS, coding gene for same and application
  • Glyphosate-resistant EPSP synthetase MC1-EPSPS, coding gene for same and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the modification synthesis of MCl-EPSPS gene

[0052] 5-enolpyruvate shikimate-3-phosphate synthase (5-enolpyrul-shikimate-3-phosphatesynthase, EPSPS) gene (sequence 4 of the sequence listing) is optimized to make it more suitable for transcription in plants, after A large number of sequences were analyzed and optimized, and a gene with the best effect was obtained through screening, named MC1-EPSPS gene. The MC1-EPSPS gene is shown in sequence 2 of the sequence listing, and encodes the protein shown in sequence 1 of the sequence listing.

Embodiment 2

[0053] Embodiment 2, glyphosate herbicide tolerance experiment

[0054] 1. Use the double-stranded DNA molecule shown in Sequence 2 of the sequence listing to replace the fragment between the NdeI and Xho restriction sites of the PET30a(+) plasmid to obtain the recombinant vector PET30a-MC1-EPSPS (sequencing verification).

[0055] 2. Introduce the recombinant vector obtained in step 1 into ER2799 competent cells to obtain recombinant bacteria ER2799-MC1-EPSPS.

[0056] 3. Inoculate the recombinant bacteria obtained in step 2 into LB liquid medium containing 50mg / L kanamycin, and culture at 37°C with constant temperature and 200rpm shaking until OD 600nm =0.5, collect the bacteria by centrifugation, resuspend the bacteria in M9 liquid medium, and adjust the OD of the bacteria solution 600nm =0.5, the bacterial suspension was obtained.

[0057] 4. After completing step 3, take 1ml of the bacterial suspension and inoculate it into 100ml of M9 liquid medium containing different...

Embodiment 3

[0064] Embodiment 3, the construction of glyphosate-resistant gene MC1-EPSPS plant expression vector

[0065] The double-stranded DNA molecule shown in Sequence 3 of the sequence table was inserted into the SmaI restriction site of the plant expression vector pTF101.1 to obtain the eukaryotic recombinant expression vector pTF101.1-MC1-EPSPS-Bar (sequencing verification). In the sequence 3 of the sequence listing, the 1-1403 position from the 5' end is the P-ract1 promoter, the 1416-1640 position is CTP2 (Arabidopsis thaliana chloroplast transfer peptide), and the 1641-3008 position is the MC1-EPSPS gene, Positions 3009-3282 are NOS terminators. The promoter in the eukaryotic recombinant expression vector pTF101.1-MC1-EPSPS-Bar that initiates the transcription of the MC1-EPSPS gene is the P-ract1 promoter, and the marker gene is the herbicide-resistant gene Bar of Streptomyces hygroscopicus.

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Abstract

The invention discloses glyphosate-resistant EPSP synthetase MC1-EPSPS, a coding gene for the same and application. The glyphosate-resistant EPSP synthetase MC1-EPSPS, the coding gene and the application have the advantages that expression of artificially modified synthetic glyphosate-resistant gene MC1-EPSPS sequences in plants can be greatly enhanced as compared with original EPSPS gene sequences; glyphosate-resistant genes MC1-EPSPS can be efficiently and stably expressed in plant cells; stable genetic MC1-EPSPS transformants can be obtained after the glyphosate-resistant genes MC1-EPSPS are introduced into tobaccos and corn, and accordingly the glyphosate resistance of the plants can be improved; crops such as soybeans, cotton, paddy rice and vegetables can be transformed by the aid ofthe genes, accordingly, the crops can have corresponding glyphosate-resistant activity and are applicable to minimum-tillage and non-tillage systems, water retention can be reinforced, soil loss canbe reduced to a great extent, and the glyphosate-resistant EPSP synthetase MC1-EPSPS and the coding gene have important economic value and broad application prospects.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a glyphosate-resistant EPSP synthetase M1-EPSPS and its coding gene and application. Background technique [0002] Glyphosate is a non-selective systemic conduction, broad-spectrum herbicide, which has the advantages of physical and chemical properties, stable quality, high efficiency, low toxicity, low residue, easy to be decomposed by microorganisms, and does not damage the soil environment. It has been widely used in In agricultural production, it has become the largest pesticide variety in the world. The relevant mechanism of action is that glyphosate is an analog of phosphoenolpyruvate (PEP), so glyphosate, shikimate-3-phosphate, and EPSP synthetase are easily combined to form EPSP-S3P-glyphosate complexes (This complex is very stable), preventing the combination of PEP and EPSP synthase, so that glyphosate competitively inhibits the activity of 5-enol-pyruvylshikim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/62A01H5/00A01H6/82
CPCC07K14/415C07K2319/00C12N9/1092C12N15/8275C12Y205/01019
Inventor 谢树章杨小艳雷开荣翁建峰王忠伟
Owner CHONGQING ACAD OF AGRI SCI