Expression,Characterization, and Site-Directed Mutation of a Multiple Herbicide-Resistant Acetohydroxyacid Synthase (rAHAS) from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Lm10 |
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Authors: | Zhi-Fei Lang Jing-Jing Shen Shu Cai Jun Zhang Jian He Shun-Peng Li |
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Institution: | (1) College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People’s Republic of China;(2) Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing, 210095, Jiangsu, People’s Republic of China; |
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Abstract: | A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas
putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W]
were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher
resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold,
6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used
as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K
m value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed
mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134,
S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred
resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four
herbicides. |
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