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Glyphosate acetyl transferase gene and its application

A glyphosate, transgenic plant technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as loss of herbicide activity

Active Publication Date: 2006-05-17
LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Because glyphosate can be absorbed by plants, and may accumulate in some meristems that affect plant growth and yield (W.A.Pline, J.W.Wilcut, S.O.Duke, K.L.Edmisten, R.Wells, J.Agric.Food Chem. 50, 506(2002)), so the use of glyphosate has potential disadvantages to people
[0007] Glyphosate can be effectively detoxified by N-acetylation, thereby losing herbicide activity, and acetylated glyphosate is not an effective substrate for EPSPS

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Construction of a total DNA gene library in glyphosate-contaminated soil

[0023] 1. Extraction of soil microbial total DNA (method with reference to Acta Microbiology, April 2003, vol.43, No.2)

[0024] 1) Weigh 1 gram of soil sample, add 3ml of DNA extraction solution and mix well, then add 20ul proteinase K (10mg / ml), incubate at 37°C for 30 minutes, mix well in between.

[0025] 2) Add 0.3ml of 20% SDS, bathe in water at 65°C for 2 hours, mix by inverting every 15-20 minutes.

[0026] 3) Freeze at -70°C, then melt at 65°C, repeat 3 times.

[0027] 4) Centrifuge at 6000xg for 10 minutes at room temperature, collect the supernatant in a new 10ml centrifuge tube

[0028] 5) Add 1ml of DNA extraction solution and 0.1ml of 20% SDS to the precipitation, vortex for 10s, bathe in water at 65°C for 10 minutes, centrifuge at 6000xg at room temperature for 10 minutes, and combine the supernatants.

[0029] 6) Repeat step 4 to combine the supernatants.

[0030] 7)...

Embodiment 2

[0060] Example 2 Method for functional screening of glyphosate acetyltransferase gene

[0061] 1. the transformed bacterium is coated on the LB solid medium that adds X-gal, IPTG and Amp, obtains about 10,000 white recombinant sub-colonies after 16 hours, then they are transferred to add 20mM glyphosate and Amp ( 60 μg / ml) on the M9 solid medium, after 72 hours, it was observed that the colony capable of growing was a clone containing the glyphosate acetyltransferase gene. Plasmids were extracted from positive clones, verified by enzyme digestion, and sequenced.

[0062] 2. Extract plasmid DNA by alkaline lysis:

[0063] 1) Inoculate the selected positive clone colony into 5ml liquid LB medium, shake at 200rpm at 37°C overnight

[0064] 2) Take 1.5ml of the culture solution in an Eppendorf centrifuge tube and centrifuge at 12,000rpm for 2min.

[0065] 3) Discard the supernatant completely, suspend and precipitate the bacterial cells with 100 μl solution I (50 mmol / L sucrose...

Embodiment 3

[0073] Example 3 The sequence of glyphosate acetyltransferase gene

[0074] A positive recombinant was screened from the gene library, and the enzyme digestion results showed that a fragment of about 1Kb was inserted. Sequencing analysis found that it contained a 441bp ORF sequence, which was similar to the glyphosate acetyltransferase gene with known functional activity (Genebank accession) No. AY597417) nucleotide sequence homology: 74.60%.

[0075] DNA nucleotide sequencing was completed by Shanghai Sangon Biotech Co., Ltd. The nucleotide and amino acid analysis software was mainly DNAman and the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih .gov / ) for the Blast program.

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PUM

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Abstract

The present invention relates to glyphosate N-acetyltransferase (GAT) gene and its application. The present invention screens out one new kind of GAT gene, and introduces the gene into receptor bacterium E. coli BL21 and type plant tobacco to make the gene engineering bacterium strain and the transgenic tobacco obtain raised tolerance on herbicide glyphosate.

Description

Technical field: [0001] The invention relates to a glyphosate acetyltransferase gene, and also relates to a plasmid containing the glyphosate acetyltransferase gene, and the use of the gene in preparing transgenic engineering bacteria and cultivating transgenic plants. Background technique: [0002] Glyphosate (glyphosate) is a broad-spectrum killing, systemic excellent herbicide, and one of the most widely used herbicides in the world (Ren Bufan, Cao Songseng. 1998. Glyphosate and Research Progress. Pesticides .37(7):1-8). [0003] However, glyphosate is a non-selective herbicide that also kills crops. Therefore, at present, it is often only applied in orchards, rubber gardens, and non-cultivated land, or applied to pre-sowing or post-sowing treatment of corn, soybeans, and cotton in no-till land, and directional treatment after emergence. This greatly limits the application scope and application time of glyphosate as a herbicide. [0004] In order to use glyphosate in t...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/63C12N15/70C12N15/82
Inventor 林敏顿宝庆陆伟金丹
Owner LONGPING BIOTECHNOLOGY (HAINAN) CO LTD
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