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Cloning, Expression and Characteristics of the Acetohydroxyacid Synthase (AHAS) Gene from Pseudomonas sp. L19(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

Issue:
2012 01
Page:
49-54
Research Field:
Articles
Publishing date:

Info

Title:
Cloning, Expression and Characteristics of the Acetohydroxyacid Synthase (AHAS) Gene from Pseudomonas sp. L19
Author(s):
LANG Zhifei YU Xinyan HUANG Xing ZHANG Juan HE Jian
(1College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China)
(2Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China)
Keywords:
acetohydroxyacid synthase inhibitor herbicide-resistant acetohydroxyacid synthase (AHAS) Pseudomonas enzymatic characteristics
CLC:
Q936 : Q78 : S482.4
PACS:
DOI:
10.3724/SP.J.1145.2012.00049
DocumentCode:

Abstract:
Acetohydroxyacid synthase (AHAS) is the target of AHAS-inhibitor herbicides, such as sulfonylurea, imidazolinone, triazolopyrimidine sulfonamide and pyrimidinyl oxybenzoate. It was recognized that AHAS herbicide-resistant mutant has potential application in the construction of transgenetic herbicide-resistant crop and the development of new potent herbicidal compounds. In this study, an AHAS inhibitor-resistant bacterium L19 was isolated from farm soil treated with metsulfuron-methyl for many years. The strain was preliminarily identified as Pseudomonas sp. according to its morphological, physiological and biochemical properties and the homology analysis of its 16S rDNA sequence. The gene coding for acetohydroxyacid synthase was cloned from Pseudomonas sp. L19 by PCR technique. The AHAS regulatory subunit of Pseudomonas sp. L19 was identical to that of P. putida KT2440, whereas four different sites were found in the catalytic subunit: [Val 29→Ala (L19→KT2440), Pro93→Ser, Val 345→Ala, Pro 484→Arg]. The AHAS of Pseudomonas sp. L19 and P. putida KT2440 were over-expressed, purified and characterized. The AHAS of Pseudomonas sp. L19 showed multiple resistance to four types of AHAS-inhibitor herbicides. The resistance factors of rAHAS were 53.6-fold, 9.3-fold, 8.2-fold and 9.5-fold to the AHAS of P. putida KT2440 when metsulfuron-methyl, imazethapyr, flumetsulam and pyriminobac-methyl used as inhibitor, respectively. The specific activity, Kc for ThDP and Mg2+ of Pseudomonas sp. L19 AHAS were lower than that of P. putida KT2440 AHAS, while the Km value of Pseudomonas sp. L19 AHAS for pyruvate was approximately 1-fold higher than the corresponding value of P. putida KT2440. Fig 1, Ref 4, Ref 30

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