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Ammonia phosphine oxidoreductase with glyphosate degradation activity and application of ammonia phosphine oxidoreductase

A reductase and phosphine oxidation technology, applied in the field of genetic engineering, can solve the problems of low screening efficiency, time-consuming and laborious

Inactive Publication Date: 2020-09-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is easily restricted by experimental materials, and a stable screening method must be established. The overall screening efficiency is low and time-consuming.

Method used

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  • Ammonia phosphine oxidoreductase with glyphosate degradation activity and application of ammonia phosphine oxidoreductase
  • Ammonia phosphine oxidoreductase with glyphosate degradation activity and application of ammonia phosphine oxidoreductase
  • Ammonia phosphine oxidoreductase with glyphosate degradation activity and application of ammonia phosphine oxidoreductase

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Cloning of glyphosate degradation gene APOX3

[0040] The invention retrieves and analyzes whether there are enzymes metabolizing aminophosphonic acid compounds through biological information, and verifies whether such enzymes have degrading activity to glyphosate. Specifically, we used aminophosphonate as the keyword to search the protein classification database InterPro (http: / / www.ebi.ac.uk / interpro / search / text / ), and the feedback result was a putative aminophosphonate oxidoreductase (Putative aminophosphonate oxidoreductase, accession number: IPR017715). The putative aminophosphonate oxidoreductase (hereinafter abbreviated as APOX) is a FAD (riboflavin adenine dinucleotide)-dependent oxidoreductase, belonging to the oxidoreductase of the pfam01266 gene family, widely distributed in pseudomonas Bacteria, Arthrobacter and other microorganisms. In this family of enzymes, 3-phosphate glycerol dehydrogenase, sarcosine oxidase β subunit and some amino acid dea...

Embodiment 2

[0060] Example 2 Determination of Recombinant Escherichia coli Glyphosate Resistance

[0061] Add the M9 solid medium culture of glyphosate: prepare the M9 plate containing 50, 100, 200mM glyphosate respectively (recipe: Na 2 HPO 4 6.8g, KH 2 PO 4 3g, NaCl 0.5g, NH 4 Cl 1g, 20mL 1mol / L glucose, 2mL 1mol / LMgSO 4 , 100μL 1mol / L CaCl 2 Add double distilled water to 1 L, adjust pH to 7.6 before sterilization, autoclave at 121°C for 30 minutes, add 2% agar), and autoclave at 121°C for 30 minutes.

[0062] Use recombinant Escherichia coli DH5α, inoculate into Ampicillin-resistant LB, culture overnight at 37°C, collect the bacteria and wash to remove the LB medium, resuspend with different volumes of sterile water to adjust the OD 600 To 1.0, take 3 μL of the washed bacterial liquid and add it dropwise to the glyphosate M9 plate, inoculate the bacterial liquid three times at different positions on each group of plates, and after the bacterial liquid is air-dried, culture it u...

Embodiment 3

[0063] The expression of embodiment 3 protein

[0064]Transform the pGEX-6p-APOX3 recombinant plasmid into the E. coli protein expression host BL21(DE3): take 1 μL of the purified pGEX-6p-APOX3 recombinant plasmid and mix it with 50 μL of E. coli competent cells DE3, and add it to a pre-cooled 1mm electroporation cuvette ( (purchased from Bio Rad Company), electric shock at 2.1KV; quickly add 500 μL of anti-antibiotic liquid LB medium, and resume culture at 37 ° C for 60 min; spread on LB plate containing 100 μg / mL ampicillin (Ampicillin, purchased from Beijing Transgene Company) overnight at 37°C, and the positive transformant verification protocol was the same as in Example 1.

[0065] Inoculate the correctly identified recombinant Escherichia coli into 10ml liquid LB medium containing 100μg / mL ampicillin, then transfer to 10mL ampicillin LB liquid medium according to the inoculation amount of 1% volume ratio, and cultivate at 37°C for 5-6h, until OD 600 When it reaches 0....

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Abstract

The invention belongs to the technical field of gene engineering, particularly relates to ammonia phosphine oxidoreductase with glyphosate degradation capacity, and further discloses application of the ammonia phosphine oxidoreductase and an encoding gene thereof in the fields of cultivation of novel glyphosate-resistant crops and glyphosate pollution biodegradation. The ammonia phosphine oxidoreductase with glyphosate degradation capability has a brand-new glyphosate decomposition mechanism, a C-N bond connecting glycine and phosphorylmethyl in glyphosate can be specifically cut, and then glycine and other non-toxic products are produced. By introducing the enzyme encoding gene into escherichia coli cells, the escherichia coli cells can be endowed with high resistance to glyphosate. The enzyme and the encoding gene of the enzyme have great application potential in degradation and restoration of glyphosate pollution or cultivation and research of glyphosate-resistant plants based on genetic engineering.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, specifically relates to an ammoniaphosphine oxidoreductase with glyphosate-degrading ability, and further discloses that the ammoniaphosphine oxidoreductase and its coding gene are used in the cultivation of new glyphosate-resistant crops and grasses. Application in the field of glyphosate pollution biodegradation. Background technique [0002] Glyphosate (trade name Roundup), also known as N-(phosphonomethyl)glycine (GLP), is a target for 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS ) is a non-selective broad-spectrum high-efficiency herbicide, which blocks the synthesis of plant aromatic amino acids by inhibiting EPSPS activity, and eventually causes plant death. The slow action of the herbicide and the fluidity of the phloem enable it to be fully transported to various parts of the plant and kill all meristems, so it has a strong and stable herbicidal effect. Since its co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12N15/82A01H5/00C12R1/19
CPCC12N9/0004C12N15/8275
Inventor 吴高兵柯达林拥军华雨郭亮阮丽芳
Owner HUAZHONG AGRI UNIV
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