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A kind of glyphosate-resistant epsp synthetase mc1-epsps and its coding gene and application

A technology that integrates genes and genes, applied in applications, genetic engineering, enzymes, etc., can solve problems such as low expression efficiency

Active Publication Date: 2021-08-13
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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  • A kind of glyphosate-resistant epsp synthetase mc1-epsps and its coding gene and application
  • A kind of glyphosate-resistant epsp synthetase mc1-epsps and its coding gene and application
  • A kind of glyphosate-resistant epsp synthetase mc1-epsps and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the transformation and synthesis of MC1-EPSPS gene

[0052] The 5-enolpyrul-shikimate-3-phosphate synthase (5-enolpyrul-shikimate-3-phosphate synthase, EPSPS) gene (sequence 4 of the sequence listing) was optimized to make it more suitable for transcription in plants. After a lot of sequence analysis and optimization, a gene with the best effect was obtained by screening, which was named MC1-EPSPS gene. The MC1-EPSPS gene is shown in SEQ ID NO: 2 of the Sequence Listing, and encodes the protein shown in SEQ ID NO: 1 of the Sequence Listing.

Embodiment 2

[0053] Example 2. Glyphosate herbicide tolerance test

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

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

[0056] 3. The recombinant bacteria obtained in step 2 were inoculated into the LB liquid medium containing 50 mg / L kanamycin, and incubated at 37°C with constant temperature and 200 rpm shaking to OD. 600nm = 0.5, centrifuge to collect the bacteria, use M9 liquid medium to resuspend the bacteria, adjust the OD of the bacteria 600nm = 0.5 to obtain a bacterial suspension.

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

Embodiment 3

[0064] Example 3. Construction of glyphosate-resistant gene MC1-EPSPS plant expression vector

[0065] The double-stranded DNA molecule shown in sequence 3 of the sequence listing 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 (which has been verified by sequencing). In Sequence 3 of the sequence listing, the 1-1403th position from the 5' end is the P-ract1 promoter, the 1416-1640th position is CTP2 (Arabidopsis chloroplast transfer peptide), and the 1641-3008th position is the MC1-EPSPS gene, Positions 3009-3282 are NOS terminators. The promoter for initiating transcription of the MC1-EPSPS gene in the eukaryotic recombinant expression vector pTF101.1-MC1-EPSPS-Bar 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 a glyphosate-resistant EPSP synthetase M1-EPSPS, its coding gene and application. Compared with the original EPSPS gene sequence, the synthetic anti-glyphosate gene MC1-EPSPS sequence of the present invention greatly enhances its expression in plants. The anti-glyphosate gene MCl-EPSPS of the present invention can be efficiently and stably expressed in plant cells. After the glyphosate-resistant gene MC1-EPSPS is introduced into tobacco and corn, stable genetic MC1-EPSPS transformants can be obtained to improve the glyphosate resistance of plants. In addition, the gene can also transform crops such as soybeans, cotton, rice, and vegetables to have corresponding glyphosate-resistant activities, so that it is suitable for low-tillage and no-tillage systems, strengthens water retention, and greatly reduces soil loss. 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 synthase MC1-EPSPS and its encoding gene and application. Background technique [0002] Glyphosate is a systemic non-selective, broad-spectrum biocidal herbicide with 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 most productive pesticide variety in the world. Its related mechanism of action is that glyphosate is an analog of phosphoenolpyruvate (PEP), so glyphosate, shikimate-3-phosphate, and EPSP synthase are easily combined to form EPSP-S3P-glyphosate complexes. (This complex is very stable), preventing the binding of PEP to EPSP synthase, so that glyphosate competitively inhibits the activity of 5-enolpyruvylshikimate-3-phosphate synthase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/62A01H5/00A01H6/82A01H6/46
CPCC07K14/415C07K2319/00C12N9/1092C12N15/8275C12Y205/01019
Inventor 谢树章杨小艳李新海雷开荣翁建峰王忠伟
Owner CHONGQING ACAD OF AGRI SCI