Glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof

An aquatic Lahnella anti-glyphosate technology, which can be used in applications, genetic engineering, plant genetic improvement, etc., can solve the problem of weak protein activity

Inactive Publication Date: 2012-07-11
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some glyphosate-resistant corn contains an altered EPSPS gene from corn. The EPSPS synthetase transcribed and translated from this altered gene is also not se

Method used

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  • Glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof
  • Glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof
  • Glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 strain separation

[0042] Take by weighing 1g of the grape root sample 1g that is frequently used in vineyards with crown smear disease, add 0.9% (w / v) sodium chloride solution 1ml, shake and mix at 5000 rev / min, and centrifuge gently at 3000 rev / min. Pour off the supernatant, then add 1ml of 0.9% (w / v) sodium chloride solution, shake and mix at 5000 rpm, let it stand on ice for 10 minutes, draw 150μl of the solution and coat it with LB solid medium containing 60mM glyphosate Cultured in medium for 24h. 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 solid medium containing 200mM glyphosate for 24h, and finally put Well-growing colonies were selected for further study.

[0043] Cultivate the screened bacterial strains in LB medium at 28°C for 48 hours, dilute the bacterial solution to 108-109 / mL, take 10 μl of the bacterial so...

Embodiment 2

[0044] Extraction and strain identification of embodiment 2 total DNA

[0045] The bacterium single strain that is isolated in embodiment 1 is cultivated 16 hours in 10ml liquid LB medium (5g / L yeast extract, 5g / LNaCl, 10g / L tryptone, phosphate buffer pH7.5), thalline culture The solution was centrifuged at 6,000 g for 5 min to obtain bacterial pellets. These pellets were first frozen at -20°C for 1 h, then washed once with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and then 20 μl of sterile lysozyme (Sigma-Aldrich) at a concentration of 10 mg / mL was added. Suspend in water and incubate on a shaking table at 37 °C for 1 h.

[0046] Then add 50 μl of 0.5M EDTA, 50 μl of 10% (w / v) SDS and 50 μl of 5M NaCl and shake gently to mix, then add 10 μl of 20 mg / mL protein kinase K (Takara Japan) , and the reaction was incubated at 37°C for 1 h. DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1, volume ratio) equivalent to the volume of the culture liquid (1 volume...

Embodiment 3

[0049] Example 3 Isolation and sequence analysis of glyphosate-resistant DNA fragments

[0050] Use different concentrations (0.02-0.5u / μL) of restriction endonuclease Sau3A (Bao Biological Engineering (Dalian) Co., Ltd.) to digest the total DNA of Lahnella aquatica obtained in Example 2 for 20-60 minutes, Then 0.7% (w / v) agarose gel electrophoresis, excise the DNA fragment with a length of 5-7kb, and recover it with a gel kit (Shanghai Sangon Bioengineering Co., Ltd.).

[0051] Select pG251 (CN1338515NCBI) as the expression vector, digest with restriction endonuclease BamHI (Bao Biological Engineering (Dalian) Co., Ltd.) and perform gel recovery.

[0052] First, dephosphorylate the digested vector plasmid to reduce the self-ligation of the plasmid, and inactivate alkaline phosphatase (Sambrook, Molecular Cloning Handbook, 1989), and then combine with the exogenous DNA fragment (ie length A 5-7kb Pantoea agglomerans DNA fragment) connection. Before ligation, use the method o...

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Abstract

The invention relates to a glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof, particularly to a gene coded as 5- enol acetone shikimic acid-3-EPSP synthase of the rahnella aquatilis having antagonistic function against the grape crown gall from the vineyard through function complementation method, and the application to the improvement of the resistance of the plant transformed from the gene to the glyphosate herbicide.

Description

technical field [0001] The invention belongs to the field of microbes, and relates to a glyphosate-resistant herbicide gene AroA-Ra derived from grape crown gall antagonistic bacteria Lahnella aquatica and its application, in particular to a method of using functional complementarity to extract the herbicide gene AroA-Ra from vineyards The 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase) gene (AroA-Ra) encoded by rahnella aquatilis which is antagonistic to grape crown gall, and the transformation of this gene into Application of plant rearing in increasing plant resistance to glyphosate herbicides. Background technique [0002] The selectivity of herbicides to destroy weeds without harming crops is a difficult property to obtain because the plant physiological and biochemical processes affected by herbicides, such as photosynthesis and amino acid biosynthesis, are common to crops and weeds. Crop cultivars and related species usually do not have the characteristic...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/82
Inventor 姚泉洪彭日荷熊爱生付晓燕田永生赵伟金晓芬韩红娟陈晨
Owner SHANGHAI ACAD OF AGRI SCI
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