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Double mutant of glyphosate-tolerant plant-derived 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and clone, expression and application of double mutant

A glyphosate-resistant, double-mutation technology, applied in the field of genetic engineering, can solve the problems of glyphosate-resistant performance not outstanding, sensitivity, etc.

Active Publication Date: 2020-05-12
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The currently used resistance genes are all EPSPS derived from Agrobacterium tumefaciens CP4, but Brassica napus EPSPS is sensitive to glyphosate, and its resistance to glyphosate is not outstanding

Method used

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  • Double mutant of glyphosate-tolerant plant-derived 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and clone, expression and application of double mutant
  • Double mutant of glyphosate-tolerant plant-derived 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and clone, expression and application of double mutant
  • Double mutant of glyphosate-tolerant plant-derived 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and clone, expression and application of double mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 BnEPSPS saturation mutant library construction and screening

[0035] According to the EPSPS conservative sequence LFLGNAGTAMRPL, the positions corresponding to the double mutation T102 / P106 in BnEPSPS were found, which were respectively located at positions 167 and 171 of BnEPSPS, and the primers were designed as follows:

[0036] BnS-F:

[0037] 5'-NNNACGCATGGCNNNTCCAGCATTCCCAAGGTACAACTCGATATCACTC-3';

[0038] BnS-R:

[0039] 5'-GTACCTTGGGAATGCTGGANNNGCCATGCGTNNNCTTACCGCTGCAG-3';

[0040] N in the primer represents a mixture of four bases A / G / C / T, which was synthesized by Wuhan Jinkairui Biotechnology Co., Ltd.

[0041] Saturation mutation PCR was obtained by pGEX-6p-BnEPSPS (the coding gene was recombined on the pGEX-6p-1 plasmid vector, the spectrum is shown in the attached figure 2 Shown) the recombinant plasmid was used as a template, and circular PCR was performed using saturated mutation primers. The PCR reaction system is shown in Table 1 below. ...

Embodiment 2

[0053] Embodiment 2 mutant BnEV1 and BnEV2 resistance analysis

[0054] The coding genes of the obtained double mutants BnEV1 and BnEV2 were respectively recombined on the pGEX-6p-1 plasmid vector to obtain pGEX-6p-BnEV1 and pGEX-6p-BnEV2 plasmids.

[0055] Transform pGEX-6p-BnEPSPS, pGEX-6p-BnEV1, and pGEX-6p-BnEV2 plasmids into E.coliAB2829 competent, pick a single colony of the transformant and place it in ampicillin-resistant LB, at 37°C Cultivate overnight, wash 2 times to remove LB nutrient components, adjust the bacterium concentration OD600 to 1.0, transfer in the M9 basal salt medium containing different concentrations (0mM, 50mM, 100mM, 200mM) glyphosate by 2% transfer amount, Set up 3 parallels for each group. Cultivate on a shaker at 37°C and 200r / min, and measure the OD600 value with a spectrophotometer every 3h. Determine the growth curve as image 3 As shown, it can be seen that the resistance levels of double mutants BnEV1 and BnEV2 are significantly improve...

Embodiment 3

[0056] Embodiment 3 Mutant BnEV1 expression and purification

[0057] Transform pGEX-6p-BnEV1 and pGEX-6p-BnEPSPS plasmids into E.coli BL21(DE3) respectively, activate the identified positive clones overnight, transfer to 10mL containing 100μg / mL Ampicillin with a volume ratio of 5% In LB liquid medium, culture at 37°C for 3h-5h until OD600 reaches about 0.6, add inducer IPTG to make the final concentration to 0.1mM, induce culture at 18°C ​​and 180r / min for 16h. The cells were collected by centrifugation, and then washed twice with Hepes buffer (50 mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), adjusted to pH 7.6, supplemented with double distilled water to 1 L), and then buffered with 1 mL of Hepes After suspending, the cells were disrupted with a sonicator. The cell lysate was centrifuged at 12,000 r / min at 4°C for 10 min, and the supernatant and precipitate were collected respectively. The precipitate was washed twice with Hepes buffer and suspended in 100 μL of ...

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Abstract

The invention belongs to the technical field of gene engineering, in particular to a double mutant obtained by performing directed evolution on 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) derived from cabbage type rape through a gene mutation technique to improve glyphosate tolerance of the EPSPS, and discloses the double mutant and application of a coding gene of the double mutant in thefield of cultivation of novel glyphosate-tolerant crops. A gene BnEPSPS of the EPSPS derived from the cabbage type rape is modified through an in vitro directed evolution technique to make the glyphosate tolerance of the EPSPS improved to further obtain the double mutant BnEV1 and BnEV2 of the glyphosate-tolerant plant-derived EPSPS. Enzymatic property determination of the mutant BnEV1 and BnEV2 shows that the glyphosate tolerance is obviously promoted, and verifies that the resistance of a plant is improved by applying the double mutant to the plant; an important genetic resource can be provided for cultivation of a transgenic glyphosate-tolerant new rape variety which is easily accepted by consumers; and the double mutant has extensive application value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method of directional evolution of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) derived from Brassica napus through gene mutation technology, so that its glyphosate Resistance is improved, and the application of the mutant and its coding gene in the field of breeding new glyphosate-resistant crops is further disclosed. Background technique [0002] When crops are planted, because weeds compete with crops for limited light, water and nutrients, the yield of crops will be seriously affected, and the increasing cost of manual weeding will also reduce crop income. The chemical name of glyphosate (trade name: Roundup) is N-(phosphonomethyl)glycine (GLP). Glyphosate has a similar structure to glycine and is a derivative of glycine. Glyphosate is a non-selective, high-efficiency herbicide with the characteristics of high efficiency, broad-spectrum and e...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/82A01H5/12A01H6/82
CPCC12N9/1092C12N15/8275C12Y205/01019
Inventor 吴高兵吴方曾玉兰林拥军郭亮阮丽芳
Owner HUAZHONG AGRI UNIV
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