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Mutant glyphosate degrading enzyme and cloning, expression and application thereof

A glyphosate and enzyme-degrading technology, applied in the field of molecular biology, can solve problems such as food threats, glyphosate activity reduction, and limited application development, achieving efficient decomposition, large application potential, and the effect of solving residual problems

Inactive Publication Date: 2019-09-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the above two strategies for implementing glyphosate tolerance in crops cannot decompose glyphosate in plants, and the residue of glyphosate in plants will bring adverse effects on the ecological environment, and the grasses transferred to seeds Glyphosate poses a threat to food
GO can catalyze the degradation of glyphosate, but its natural substrate is glycine, which reduces the activity of glyphosate. In theory, it will affect the metabolism of plant glycine after being introduced into plants, which limits the application and development of GO.

Method used

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  • Mutant glyphosate degrading enzyme and cloning, expression and application thereof
  • Mutant glyphosate degrading enzyme and cloning, expression and application thereof
  • Mutant glyphosate degrading enzyme and cloning, expression and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Study on site-directed mutation of the 63rd amino acid of glyphosate degrading enzyme

[0028] With the recombinant plasmid pGEX-6p-B4S7 (see the attached figure 1 (shown) is the template (pre-laboratory construction, reference Int J Biol Macromol, 2015, 79: 965-970), using the designed circular expansion primer (see Table 1 below), oligonucleotide-mediated The PCR site-directed mutagenesis method (guided according to the QuikChange Site-directedMutagenesis kit kit of Stratagene Company) introduced a mutation at the 63rd amino acid residue site of glyphosate degrading enzyme, and its coding gene was passed through pGEX-6p-1 (purchased From GE Healthcare in the United States, its plasmid map is attached figure 2 Shown) on the plasmid vector and recombined into pGEX-6p-F63X plasmid.

[0029] The specific PCR reaction conditions are: pre-denaturation at 97°C for 2 min; denaturation at 95°C for 20 sec; annealing at 54°C for 30 sec; extension at 72°C for 2 min a...

Embodiment 2

[0033] Example 2 Glyphosate degrading enzyme mutant protein expression and purification

[0034] The recombinant plasmid pGEX-6p-F63X containing the mutant F63X gene (see attached image 3 shown) were mixed with E.coli BL21(DE3) competent cells, transformed by electric shock, spread on ampicillin-resistant plates (ampicillin concentration 100 μg / mL), and cultured overnight at 37°C until a single colony visible to the naked eye grew. Pick a single clone and inoculate it into 20 mL of liquid LB medium (containing 100 μg / mL of ampicillin), and culture overnight at 37° C. with shaking at 200 rpm. Transfer 1% of the bacterial solution to 2L of fresh liquid LB medium (containing ampicillin 100 μg / mL), shake culture at 37°C and 200rpm until the OD600 is 0.6, add the inducer IPTG with a final concentration of 0.1mM, and store at 22°C, 160rpm induction culture for 12h to express the protein. The bacteria were collected by centrifugation, washed twice with 50mM sodium pyrophosphate bu...

Embodiment 3

[0037] Embodiment 3 glyphosate degrading enzyme mutant enzymatic kinetics assay

[0038] In the present embodiment, the enzyme activity assay of glyphosate degrading enzyme and its mutant adopts the horseradish peroxidase coupling method (Job, Marcone et al.2002), by measuring the H released by the reaction 2 o 2 The amount reflects the enzyme activity. Using o-dianisidine as a chromogenic substrate, glyphosate is oxidized and decomposed by glyphosate-degrading enzymes to produce H 2 o 2 Oxygen is released by horseradish peroxidase to oxidize o-dianisidine to show orange red, with an absorption peak at 450nm, and measure OD 450 The value was entered into the standard curve to calculate the enzyme activity.

[0039] (1) Enzyme activity assay method

[0040] Convert 1 μmoL of substrate (containing glycine or oxygen) or generate 1 μmoL of product H at 25°C and optimal pH 2 o 2 The required enzyme amount of glyphosate-degrading enzyme is defined as one enzyme activity unit ...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a glyphosate degrading enzyme mutant which is obtained by a site-directed mutation technology and has improved catalytic activity and substrate specificity on glyphosate. According to the glyphosate degrading enzyme mutant B4S7-F63X disclosed by the invention, on the basis of an existing glyphosate degrading enzyme B4S7, the modified glyphosate degrading enzyme is mutated into alanine or histidine at the 63rd-position phenylalanine. Compared with the existing literature reports and patent data, the B4S7-F63A has the highest catalytic efficiency on glyphosate, the B4S7-F63H has the highest substrate specificity on glyphosate. After being introduced into a plant, the glyphosate degrading enzyme coding gene is expected to endow the plant with glyphosate resistance, to realize high-efficiency decomposition of the glyphosate, and to solve the problem of residue of the glyphosate in the plant. The product has great application potential in the fields of cultivating novel glyphosate-resistant crops and cultivating novel herbicide-resistant plants capable of degrading glyphosate.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a glyphosate-degrading enzyme mutant obtained by a site-directed mutation technique with improved glyphosate catalytic activity and substrate specificity, and further discloses the mutant and its coding The application of genes in the cultivation of new glyphosate-resistant crops and the biodegradation of glyphosate pollution. Background technique [0002] Glyphosate (trade name Roundup) is also known as N-(phosphonomethyl)glycine (GLP), and its molecular formula is C 3 h 8 NO 5 P, with a relative molecular weight of 169.1, glyphosate has a similar structure to glycine and is a derivative of glycine. Glyphosate is a white solid, non-volatile, with a high melting point (200°C), good stability, and low solubility in water (1.2%, 25°C). Glyphosate is a non-selective, high-efficiency herbicide that is widely used around the world and is currently the most wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/82A01H5/00
CPCC12N9/0022C12N15/8275C12Y104/03019
Inventor 吴高兵刘子铎林拥军郭一鸣
Owner HUAZHONG AGRI UNIV
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