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An isolated 5-enolpyruvateshikimate-3-phosphate synthase gene

A technology of enolacetone shikiki and phosphate synthase, applied in the field of genetic engineering

Active Publication Date: 2016-03-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the AroA genes with high resistance to glyphosate have been discovered and applied for patent protection, which has caused great limitations to the practical application in my country

Method used

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  • An isolated 5-enolpyruvateshikimate-3-phosphate synthase gene
  • An isolated 5-enolpyruvateshikimate-3-phosphate synthase gene
  • An isolated 5-enolpyruvateshikimate-3-phosphate synthase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 15

[0032] Example 15-Synthesis and Cloning of Enolpyruvyl Shikimate-3-Phosphate Synthase Gene

[0033] (1) AroA I.vari Synthesis of Gene Sequence

[0034] AroA encoding EPSPS of Isoptericola Variabilis strain obtained in Genbank database I.vari Gene sequence, the nucleotide sequence 1374bp of the gene was synthesized by Nanjing GenScript and provided with recombinant plasmid pUC57-AroA I.vari ( Figure 4). Its nucleotide sequence and encoded amino acid sequence are shown in the sequence listing SEQ ID NO:1 and SEQ ID NO:2.

[0035] (2) AroA I.vari Cloning of Gene Sequences

[0036] According to AroA I.vari Sequence design primers: add a BamHI restriction site at the 5' end of the primer, add a restriction site EcoRI at the 3' end, and the DNA sequence of the primer pair is as follows:

[0037] Forward primer (5'-AroA I.vari -BamHI):5'-CGC GGATCC ATGACGCCAGCGCCCGCCAGC-3', where the underlined part is the restriction site;

[0038] Reverse primer (3'-AroA I.vari -EcoRI...

Embodiment 2

[0053] Expression, purification and analysis of embodiment 2EPSPS protein

[0054] (1) Expression of the target protein EPSPS

[0055] The recombinant plasmid pGEX-6p-1-AroA I.vari Transform into expression host cells E. coliBL21(DE3). The identified positive clones were activated overnight, transferred to 1 L LB liquid medium containing 100 μg / mL Ampicillin at an inoculation volume of 1% by volume, and cultured at 37°C for 2-3 hours until OD 600 When it reaches about 0.6, add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5mM, and culture at 22°C and 180rpm for 25h. Collect the bacteria by centrifugation, then adjust the pH to 7.0 with Hepes buffer (recipe: 50mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), 2mM dithiothreitol (DTT), make up double-distilled water to 1L, set aside ) to wash the cells once, suspend them with 50 mL of Hepes buffer, and break the cells with a high-pressure cell disruptor (purchased from GEANiroSoari...

Embodiment 3

[0075] Example 3: Genetic transformation experiment (functional complementation verification)

[0076] The recombinant plasmid pGEX-6p-1-AroA J.limo Transformed into AroA gene-deficient Escherichia coli competent cells (for the transformation process, refer to the transformation process in the above-mentioned Example 1. This bacterial strain does not contain the AroA gene, which can eliminate the interference of the AroA gene contained in general Escherichia coli) (VaithanomsatandBrown2007), respectively Received M9 liquid medium containing 0mM, 50mM, 100mM, 150mM glyphosate (recipe: Na2HPO46.8g / L, KH2PO43.0g / L, NaCl0.5g / L, NH4Cl1.0g / L, MgSO4 7H2O0.4929g / L, CaCl20.111g / L, glucose 4.0g / L, supplemented with double distilled water to 1L, adjusted the pH to 7.4 before sterilization, and sterilized under high pressure steam at 115°C for 10min), and measured the growth curve.

[0077] The constructed recombinant plasmid pGEX-6p-1-AroA I.vari Transform into competent cells Escheri...

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Abstract

The invention relates to separation and identification of roundup ready 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene, and belongs to the technical field of agricultural microbial genetic engineering. The 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene is characterized in that the nucleotide sequence is shown as SEQ ID No.: 1; and the sequence of protein coded by the gene is shown as SEQ ID No.: 2. The 5-enolpyruvyl shikimate-3-phosphate synthase gene (EPSPS) gene is prepared by synthesizing aroA gene sequences of actinobacteria Isoptericola Variabilis as reported in Genbank; and recombinant escherichia coli LZD002 containing the gene plasmid is stored in the China Center for Type Culture Collection (CCTCC), with a storage number of CCTCC No.: M2013632. The biological experiment verifies that the gene shows a certain tolerance for glyphosate.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the isolation of a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene derived from the genome of actinomycete bacteria, whose coding gene is AroA, through gene recombination and genetic transformation The method is to transfer the gene into the host cell, so that the host cell can acquire the ability to tolerate glyphosate. The present invention relates to the nucleotide sequence and amino acid sequence of the gene. Background technique [0002] Glyphosate is the most widely used broad-spectrum transmissive herbicide in the world, and it is destructive to most plants. Glyphosate has stable physical and chemical properties, low toxicity to humans and livestock, and low residue in soil, so it can be widely used. [0003] The shikimate pathway is a key metabolic pathway in bacteria, fungi, algae and higher plants, and is closely related to the synthesis of aroma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N1/21A01H5/00C12R1/19
Inventor 刘子铎易沭远林拥军张利莉吴高兵
Owner HUAZHONG AGRI UNIV
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