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Achromobacter xylosoxidans glutamine synthetase gene and application thereof

A colorless bacteria, glutamine technology, applied in the application, genetic engineering, enzymes and other directions, can solve the problem of reducing the resistance of transgenic crops glufosinate-ammonium

Active Publication Date: 2017-12-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since only these two genes are currently used in the cultivation of glufosinate-resistant transgenic crops, it is easy to cause concerns about the reduction of glufosinate-ammonium resistance of transgenic crops in field management.

Method used

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  • Achromobacter xylosoxidans glutamine synthetase gene and application thereof
  • Achromobacter xylosoxidans glutamine synthetase gene and application thereof
  • Achromobacter xylosoxidans glutamine synthetase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Acquisition of novel glufosinate-ammonium resistance gene glutamine synthetase gene

[0031] 1. Collection of soil samples

[0032] Soil samples were collected from orchards that had been used at least four times a year and had been used continuously for more than ten years.

[0033] 2. Screening of glufosinate-resistant strains

[0034] Weigh 1g of the orchard soil sample frequently used by glufosinate-ammonium, add 1ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, centrifuge gently at 3000 rpm, pour off the supernatant, and then add 0.9% (w / v) 1ml of sodium chloride solution, shake and mix at 5000 rpm, let it stand on ice for 10 minutes, draw 150 μl of the solution, spread it and culture it in LB solid medium containing 20 mM glufosinate-ammonium for 24 hours. Inoculate the grown single colony into a test tube with 1.6ml of LB liquid medium, incubate at 28°C for 48h, then draw 150μl of the culture solution and apply it again to the LB ...

Embodiment 2

[0043] Example 2 Artificial synthesis of novel glufosinate-ammonium resistance gene glutamine synthetase gene

[0044] The gene synthesis method [Nucleic Acids Research, 2004, 32, e98] artificially synthesized obtained in Example 1

[0045] Novel glutamine synthetase gene of the present invention. A total of 34 pairs of primers were designed, and the designed primers were as follows:

[0046] 1. P1: Tm=54, 60mer

[0047] GGA, TCC, ATG, GCA, AGC, CCG, AAA, GAC, GTC, CTG, AAA, CAG, ATC, GCC, GAT, AAC, GAA, GTG, AAA, TTC

[0048] 2. P2: Tm=54, 60mer

[0049] GGT,GCT,CAC,GGC,CAA,CCG,TAT,CGG,TAA,AGC,GGA,AAT,CCA,CGA,ATT,TCA,CTT,CGT,TAT,CGG

[0050] 3. P3: Tm=54, 60mer

[0051] TAC,GGT,TGG,CCG,TGA,GCA,CCA,CGT,TTC,CGT,GCC,CAC,CAC,CGC,TAT,CGA,TGA,AGA,CAA,GCT

[0052] 4. P4: Tm=54, 60mer

[0053] GCC, CGG, GAT, CGA, CGA, ACC, GTC, GAA, AGC, CTG, CCC, GCT, TTC, CAG, CTT, GTC, TTC, ATC, GAT, AGC

[0054] 5. P5: Tm=54, 60mer

[0055] ACG,GTT,CGT,CGA,TCC,CGG,GCT,GGA,AGG,GCA,TCG,AAG...

Embodiment 3

[0116] Example 3 In vitro glufosinate-ammonium resistance test

[0117] The glutamine synthetase gene obtained in Example 2 was digested with BamH I and Sac I, and then constructed into the prokaryotic expression vector pG251 to obtain the recombinant plasmid pG251- glnA A.xylosoxidans . The recombinant plasmid pG251- glnA A.xylosoxidans , smeared on M9 plate and cultivated for 48h, picked the transformant with a toothpick and inoculated it in LB liquid medium for cultivation until the concentration reached 5 × 10 3 For cells / μL, take 2μL of cell culture fluid and 1 / 5 and 1 / 25 diluted cell culture fluid to spot on M9 plates containing different concentrations of glufosinate-ammonium (0, 30 mM, 50 mM and 70 mM) Cultivate for 48h. It was subsequently observed that 50 mM glufosinate-ammonium had started to inhibit the growth of glutamine synthetase gene cells of the present invention. But when the concentration of glufosinate-ammonium is 70mM, the glutamine synthetase ge...

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Abstract

The invention discloses a glutamine synthetase gene derived from achromobacter xylosoxidans and an application of the gene. The glutamine synthetase gene comprises 1413 basic groups and 471 coded amino acids. The nucleotide sequence of the gene is as shown in SEQ ID NO 1, and the sequence of the coded amino acids is as shown in SEQ ID NO 2. The glutamine synthetase gene derived from the achromobacter xylosoxidans has high glufosinate-ammonium tolerance and can be used for cultivating glufosinate-ammonium resistant transgenic crops.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering, and specifically relates to a glutamine synthetase gene derived from Achromobacter xylosoxidans and its application. Background technique [0002] Glufosinate-ammonium (DL-phosphinotricinm) is a broad-spectrum contact herbicide. Its principle of action is to inhibit the activity of glutamine synthetase (Glutamine synthetase, GS) in plants, resulting in blocked glutamine synthesis, nitrogen metabolism disorder, ammonium ion accumulation, thereby destroying plant cell membranes, preventing plant photosynthesis and dying. Conventional crops do not have the ability to resist or degrade glufosinate, and glufosinate will also kill conventional crops if they come into direct contact. Therefore, in order to be able to selectively kill weeds when the crops are growing, the crops need to acquire the ability to resist glufosinate-ammonium or to degrade glufosinate-ammonium. [0003] So far, th...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00A01H5/00
CPCC12N9/93C12N15/8277C12Y603/01002
Inventor 田永生许晶姚泉洪彭日荷王波付晓燕韩红娟高建杰王丽娟李振军
Owner SHANGHAI ACAD OF AGRI SCI
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