Isolation of a metsulfuron-methyl-resistant bacteria and cloning and expres-sion of the acetohydroxyacid synthase genes ilvIH

Abstract:

Abstract: Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine in plant, fungi and bacteria, and also is target of the sulfony-lurea, imidazolinone, triazolopyrimidine and other acetohydroxyacid synthase inhibitor herbicides. [Objective] The pur-pose of this study is to get the resistant gene, prepare a functional bacteria with acetohydroxyacid synthase, and investi-gate the relationship between the mutation sites of the acetohydroxyacid synthase and the herbicides-resistant. [Methods] A metsulfuron-methyl-resistant bacterium Lm10 was isolated from metsulfuron-methyl contaminated soil. Acetohy-droxyacid synthase genes ilvIH was amplified from the genome DNA of strains Lm10 by PCR. The ilvI and ilvH were cloned into the bacterial expression vector pET29a(+) respectively. [Results] Strain Lm10 was identified preliminarily as Pseudomonas sp.. It can endure 14000 mmol/L metsulfuron-methyl and showed cross resistance to diffenent acetohy-droxyacid synthase inhibitor herbicids, such as chlorsulfuron, imazethapyr, flumetsulam and penoxsulam. The alignment result of the ilvIH amino acid sequence showed that ilvI of strains Lm10 differed from that of strain KT2440 by 6 sites, While the ilvH of the two strains were the same. The gene ilvI and ilvH were functional expressed in the Escherichia coli strain BL21(DE3) respectively. The expressed production pET-I displayed acetohydroxyacid synthase activity and showed re-sistance to high concentration of metsulfuron-methyl. [Conclusion] The ilvI displayed acetohydroxyacid synthase activity. The 6 different sites in ilvI of strains Lm10 probably led to herbicids resistance.