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Resistance gene ilvBN of acetohydroxyacid synthase inhibitor herbicides

A technology of acetohydroxy acid synthase and inhibitor, which is applied in the application of environmental microorganisms and agriculture, can solve the problems of limited increase in resistance, no expression of agronomic traits, narrow drug resistance spectrum, etc., and achieves the effect of preventing key points

Inactive Publication Date: 2009-06-17
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

At present, the research and application of anti-sulfonylurea gene transgenics at home and abroad are weak, and problems such as narrow drug resistance spectrum, limited range of resistance improvement, unstable or non-expression of resistance, and changes in agronomic traits of transgenic crops have not been well resolved. Solve; research on broad-spectrum, strong resistance, and stable expression of resistance gene resources and transgenic strategies in crops needs to be further strengthened

Method used

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  • Resistance gene ilvBN of acetohydroxyacid synthase inhibitor herbicides
  • Resistance gene ilvBN of acetohydroxyacid synthase inhibitor herbicides
  • Resistance gene ilvBN of acetohydroxyacid synthase inhibitor herbicides

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Embodiment Construction

[0017] 1. Cloning of acetohydroxyacid synthase inhibitor herbicide resistance gene ilvBN large and small subunits

[0018] 1.1 Extraction of total DNA of Klebsiella sp.

[0019] The total DNA of Klebsiella sp.HR9 with high purity and large molecular weight was extracted by CTAB method, dissolved in TE (pH 8.0), and stored at -20°C. For specific methods, refer to "Refined Molecular Biology Experiment Guide".

[0020] 1.2 Amplification primers of ilvBN gene:

[0021] Forward primer: 5'-CATATGCGCTTTACCGGCGCGCA-3',

[0022] Reverse primer: 5'-CTCGAGTTACTCGAAGAACACCGCAA-3'

[0023] Gene fragments were amplified from Klebsiella sp.HR9 by PCR.

[0024] Amplification system:

[0025] LA Taq (5U / μl) 0.5μl

[0026] 10×LA PCR Buffer II (Mg2+Plus) 5μl

[0027] dNTP Mixture (2.5mM each) 8μl

[0028] Template DNA 10ng

[0029] Forward primer (20μM) 1μl

[0030] Reverse primer (20μM) 1μl

[0031] Sterilized distilled water up to 50μl

[0032] PCR amplification program:

[0033] ...

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Abstract

The invention relates to an acetohydroxy acid synthase inhibitor herbicide resistant gene ilvBN belonging to the application environment microorganism and crop science field. The invention clones an acetohydroxy acid synthase gene ilvBN with broad spectrum resistance to the acetohydroxy acid synthase inhibitor herbicide. A recombinant strain BL21(DE3) crude enzyme liquid acetohydroxy acid synthase containing the gene is higher by 40 times than the enzyme activity, has very strong resistance to various acetohydroxy acid synthase inhibitor herbicides and the resistance to the metsulfuron-methyl, chlorsulfuron, imazethapyr and pyrimidine sulam improves respectively by 15 times, 16 times, 2.7 times and 2.7 times. The AHAS gene resources resisting the herbicide can provide resistance gene resource for constructing transgene crops, thoroughly solve the phytotoxicity to the non-target crop by herbicide and have very important theory and application value.

Description

technical field [0001] The invention relates to the application of acetohydroxyacid synthase inhibitor herbicide resistance gene ilvBN, which belongs to the fields of applied environmental microorganisms and agriculture. Acetohydroxyacid synthase (AHAS) is the target of acetohydroxyacid synthase inhibitor herbicides such as sulfonylureas, imidazolinones, triazole pyrimidines, sulfonamides and pyrimidine salicylic acid. AHAS mutant gene resources resistant to such herbicides can construct transgenic crops to completely solve the phytotoxicity of herbicides on non-target crops. The invention relates to an acetohydroxyacid synthase inhibitor herbicide resistance gene and a carrier and transformant containing the gene. Background technique [0002] Acetohydroxyacid synthase (acetohydroxyacid synthase, AHAS), also known as acetohydroxyacid synthase (Acetolactates synthase, ALS), is a branched-chain amino acid (valine, leucine and isoleucine) biological The key enzyme in the fir...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N1/21A01H5/00C12R1/19
Inventor 何健李顺鹏沈晶晶黄星聂志娟
Owner NANJING AGRICULTURAL UNIVERSITY
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