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Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene

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

Inactive Publication Date: 2013-07-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the glyphosate resistance genes used in production practice are mainly the above-mentioned class II AroA genes, and there are relatively few studies on class I AroA genes, and most of the class I AroA genes discovered so far are naturally sensitive to glyphosate. This kind of research is mainly based on terrestrial microorganisms, and 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 actual application in our country.

Method used

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  • Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene
  • Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene
  • Isolation of 5-enol acetone shikimic acid-3-phosphate synthase gene

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Experimental program
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Effect test

Embodiment 1

[0031] Example 1 Isolation and cloning of 5-enolpyruvylshikimate-3-phosphate synthase gene

[0032] (1) Construction and sequencing of genomic library to obtain resistance genes

[0033] The original Janibacter limosus 1C00094 strain (also known as Janibacter limosus 1C00094) with glyphosate resistance of 150mM was selected from a natural deep-sea marine bacterium screened by the Third Research Institute of the State Oceanic Administration of China (the original strain came from China The Marine Microorganism Culture Collection and Management Center, namely the Third Research Institute of the State Oceanic Administration of China, Xiamen, Fujian, China, www.mccc.org.cn), constructed the genome library of the strain according to the following steps and methods: extracting the deep-sea marine bacterium Janibacter limosus That is, the genomic DNA of the 1C00094 strain of Mirabilis spp. (see below for the specific method), was partially enzymatically digested (the restriction site...

Embodiment 2

[0059] Expression, purification and analysis of embodiment 2 EPSPS protein

[0060] (1) Expression of the target protein EPSPS

[0061] The recombinant plasmid pGEX-6p-1-AroA J.limo Transformed into expression host cell Escherichia coli BL21(DE3). Verify the positive clones, activate the positive clones identified correctly overnight, transfer to 1L LB liquid medium containing 100μg / mL Ampicillin with an inoculation volume of 1% by volume, and culture at 37°C for 2-3h until OD 600 When it reaches about 0.6, the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5mM, and cultured at 22°C and 180rpm for 25h. The bacteria were collected by centrifugation, and then the pH was adjusted to 7.0 with Hepes buffer (recipe: 50 mM 4-hydroxyethylpiperazineethanesulfonic acid (Hepes), 2 mM dithiothreitol (DTT), and double-distilled water was added to 1 L. Standby) Wash the cells once, suspend them with 50 mL of Hepes buffer, and break the cells wit...

Embodiment 3

[0090] Embodiment 3: Genetic transformation experiment (functional complementation verification)

[0091] 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 E. coli) (Vaithanomsat and Brown 2007) , were transferred to M9 liquid medium containing 0mM, 50mM, and 100mM glyphosate, respectively, and the growth curve was measured.

[0092] (1) Set comparison

[0093] Primer design and synthesis. According to the Escherichia coli E.coli K-12 AroA gene published by GENEBANK (named AroA E.coli ) nucleotide sequence (accession number: NP_415428.1), the following primer pairs were designed and synthesized:

[0094] Forward primer (5'-AroA E.coli -BamHI):

[0095] 5'-CGC GGATCC ATGGAATCCC...

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Abstract

The invention belongs to the technical field of gene engineering, and in particular relates to isolation and identification of an anti-glyphosate gene 5-enol acetone shikimic acid-3-phosphate synthase (EPSPS). The EPSPS gene has a nucleotide sequence as described in a SEQ ID NO:1, and an amino acid sequence encoding the gene is shown as a SEQ ID NO:2. The EPSPS gene is cloned from a genomic library of a deep-sea bacterium Janibacter limosus strain. A recombinant Escherichia coli LZD001 containing the plasmid of the gene is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC No:M2011493. Biological experiments prove that the gene has certain tolerance to glyphosate.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the isolation and identification of a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene derived from the genome of deep-sea marine microorganisms, whose coding gene is AroA, through the method of genetic transformation , and transfer the gene into the host cell, so that the host cell can acquire the ability of tolerance to glyphosate. The present invention relates to the nucleotide sequence and amino acid sequence of the gene. technical background [0002] N-phosphonomethylglycine, i.e. glyphosate (glyphosate), is a well-known systemic broad-spectrum herbicide with low toxicity to humans and livestock and low residue in soil, making it It is difficult for weeds to develop resistance and other characteristics. Based on these characteristics, glyphosate has been widely used all over the world. [0003] Glyphosate can inhibit the activity of an important enzyme...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N1/21C12R1/19
Inventor 刘子铎赵艳林拥军邵宗泽
Owner HUAZHONG AGRI UNIV
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