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Glutamine synthetase gene with glufosinate tolerance and application thereof

A glufosinate-ammonium resistance technology, applied in the field of genetic engineering, can solve problems such as plant death, amino acid synthesis and photosynthetic chlorophyll destruction

Inactive Publication Date: 2017-08-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PPT can inhibit all known forms of GS, and the results of the inhibition lead to the accumulation of ammonia in cells, the inhibition of amino acid synthesis and photosynthesis, and the destruction of chlorophyll; but mainly through the rapid inhibition of RuBp carboxylase / photorespiration causing plant death

Method used

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  • Glutamine synthetase gene with glufosinate tolerance and application thereof
  • Glutamine synthetase gene with glufosinate tolerance and application thereof
  • Glutamine synthetase gene with glufosinate tolerance and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1: Screening of natural bacterial strains producing glutamine synthetase

[0021] (1) Screening of natural strains producing glutamine synthetase:

[0022] The applicant's laboratory isolated nearly 200 strains of marine bacteria from seawater near Qingdao, Shandong. The marine strains preserved by the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, where the applicant is located, were placed in high-salt LB (1% peptone, 0.5% yeast Culture and 2% NaCl, pH7.0) after activation on the plate, single bacterial colonies were picked out and cultured in liquid high-salt LB medium, and after cultivating at 28°C for 36 hours, the cells were broken by ultrasonic waves (30HZ, 400W), respectively. The supernatant after fermentation and the supernatant after cell disruption were used to measure glutamine synthetase enzyme activity (the enzyme activity assay method is as follows). Through experiments, a bacterial strain with high glutamin...

Embodiment 2

[0037] Example 2: Cloning, expression and purification of exiGS gene in Escherichia coli

[0038] The specific methods and steps are as follows:

[0039] 1. Extract (Exiguobacterium sp) chromosomal DNA:

[0040] (1) After culturing the isolated Exiguobacterium sp. strain in a high-salt [sodium chloride content of 100 μl 5mol / L, see step (4)] LB liquid culture at 28°C for 24 hours, take 1.5ml of the bacterial liquid and sterilize it once Centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and collect the bacteria.

[0041] (2) Wash the bacteria twice with TE buffer (50mmol / LTris-HCl pH8.0, 10mmol / LEDTA pH8.0), then add 50μl 100μg / ml lysozyme (purchased from sigma company) to suspend the bacteria. 37°C water bath for one hour.

[0042] (3) Add 520 μl TE buffer, 30 μl 10% SDS and 3 μl 20 mg / ml proteinase K (purchased from sigma company) and mix well, then place in a water bath at 37° C. for one hour.

[0043] (4) Add 100 μl of 5 mol / L sodium chloride solution and ...

Embodiment 3

[0069] Embodiment 3: Expression and purification of glutamine synthetase:

[0070] 1. Induced expression of recombinant glutamine synthetase in Escherichia coli:

[0071] The recombinant plasmid pGEX-6P-exiGS was transformed to express purified glutamine synthetase in Escherichia coli BL21(DE3). Mix the plasmid pGEX-6P-exiGS with 100 μl of Escherichia coli competent cells BL21 (DE3), place in ice bath for 30 minutes, treat at 42°C for 90 seconds, add 800 μl of LB medium (1% tryptone, 1% Nacl, 0.5% Yeast powder, pH 7.0), incubated at 37°C for 1 hour, spread a solid LB plate containing 100 μg / ml ampicillin, incubated at 37°C for 14 hours, and picked transformants to induce the expression of glutamine synthetase.

[0072] Transfer overnight activated transformants to 1L LB liquid medium containing 100μg / ml ampicillin at 1% inoculum size, culture at 37°C for 2-3hrs, and make the OD 600 When it reaches 0.6-0.7, add IPTG to a final concentration of 0.2mM, and culture at 18°C ​​at ...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a glutamine synthetase gene with glufosinate tolerance and application thereof. The sequence and coding region of the glutamine synthetase gene exiGS with glufosinate tolerance are disclosed, the nucleotide sequence of the gene is shown in SEQ ID NO:1, and 446 amino acids are coded. The sequence of protein coded by the gene is shown in SEQ ID NO:2. Biological function verification proves that the gene is high in activity, resistant to low temperature and high in high-temperature stability. The functions and the potential application to anti-glufosinate crops of the glutamine synthetase gene with glufosinate tolerance are verified preliminarily. The gene can be widely applied to different herbicide-resistant transgenic plants.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to the isolation and identification of a glutamine synthetase gene with glufosinate-ammonium resistance. Background technique [0002] Glufosinate (Glufosinate) is a widely used organophosphorus herbicide, its active ingredient is phosphinothricin (PPT for short), L-PPT has phytotoxicity, and its chemical name is (RS)-2-amino-4-(hydroxy Methylphosphinyl) ammonium butyrate is a racemic mixture, which belongs to the biomimetic stem and leaf treatment agent. The research and development of glufosinate-ammonium is closely related to bialaphos. Bialaphos is a tripeptide natural product isolated and purified from the fermentation broth of Streptomyces hygroscopicus. Bialaphos itself has no herbicidal activity and is degraded into glufosinate-ammonium with herbicidal activity in plants. Based on this, the German AGFO company directly synthesized glufosinate (Glufosinate) and successful...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N1/21A01H5/00C12R1/19
CPCC12N9/93C12N15/8277C12Y603/01002
Inventor 刘子铎张绍伟林拥军吴高兵左武
Owner HUAZHONG AGRI UNIV
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