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A cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene

A technology of enolacetone shiki and phosphate synthase, applied in genetic engineering, plant gene improvement, enzymes and other directions, can solve the problem of lack of low temperature environmental adaptability EPSPS enzyme encoding gene and the like

Active Publication Date: 2021-07-13
HUAZHONG AGRI UNIV
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  • Description
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  • Application Information

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Problems solved by technology

However, according to the existing research, there is a lack of genes encoding EPSPS enzymes with high glyphosate resistance and low temperature environment adaptability in the world.

Method used

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  • A cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene
  • A cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene
  • A cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene

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Embodiment 1

[0034] 1. Screening of original strains

[0035] The marine bacterium (Isoptericolasp.GR1, implementing the present invention is not limited to this marine bacterium) isolated from the deep-sea sea mud (Qingdao, Shandong) preserved in the laboratory is contained in 2216 medium (peptone 10g, yeast powder) containing 20mM glyphosate 5g, beef extract 2g, NaCH 2 COOH 1g, NH 4 NO 3 0.2g, NaCl 19.45g, MgCl 2 0.5g, MgSO 4 0.5g, CaCl 2 0.5g, KCl 0.55g, NaHCO 3 0.16g, add 18g of agar powder, add double distilled water to 1L, adjust the pH to 7.6 before sterilization, and sterilize under high pressure steam at 121°C for 30min; The purified single bacterium colony obtained after the rescreening was cultivated in the 2216 medium containing 500mM glyphosate, and a bacterial strain ( figure 2 ).

[0036] 2. Bacterial Genomic DNA Extraction

[0037] Cultivate marine bacteria (Isoptericola sp.GR1) with 300mM glyphosate 2216 liquid medium, cultivate overnight at 28°C, and use the...

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Abstract

The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene. The nucleotide sequence of the type I 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene is as described in the sequence listing SEQ ID NO: 1, and the protein sequence encoded by the gene is as SEQ ID NO: 2 mentioned. The cold-adapted type I 5-enolpyruvyl shikimic acid-3-phosphate synthase gene was cloned according to the conserved sequence of the aroA gene of the halophilic termite fungus Isoptericola sp.GR1TH_0001 reported in Genbank, and verified by biological experiments , proving that the enzyme has high tolerance to glyphosate and has unique low temperature adaptability.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and in particular relates to an isolated cold-adapted type I 5-enolpyruvylshikimate-3-phosphate synthase gene. The present invention isolates and identifies resistance to glyphosate-derived 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene derived from microorganisms, proving that the cloned gene of the present invention encodes 5 -The nucleotide sequence of enolpyruvylshikimate-3-phosphate synthase has higher resistance to glyphosate, and Escherichia coli cells containing the gene can tolerate glyphosate at a concentration of 200mM / L. At the same time, the enzyme still has high activity at 10°C. This enzyme is the first identified low-temperature adaptable and glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase, and its encoding gene It has great application potential in cultivating overwintering herbicide-resistant crops. Background technique [0002]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10A01H5/00A01H6/00
CPCC12N9/1092C12N15/8275C12Y205/01019
Inventor 吴高兵郭亮吴方阮丽芳刘子铎
Owner HUAZHONG AGRI UNIV
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