Isolation Of A Metsulfuron-Methyl-Resistant Bacteria And Cloning And Expression Of The Ahas IlvIH

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 sulfonylurea, imidazolinone, triazolopyrimidine and other AHAS inhibitor herbicides.A metsulfuron-methyl-resistant bacterium Lm10 was isolated from metsulfuron-methyl contaminated soil. Strain Lm10 can endure 14,000μmol/L . Based on the morphology, physiological and biochemical characteristics, and the homology analysis of its 16S rDNA sequence, Lm10 was identified preliminarily as Pseudomonas sp..The optimal growth temperature and initial pH are 30℃, 6.5 respectively. The aeration had little effect on the growth of Lm10. The optimal medium for the growth of strain Lm10 was amylum as carbon source and organic nitrogen as nitrogen source. Lm10 has resistance to a few antibiotics, such as Amp,Tc and Str. It showed cross resistance to diffenent ALS inhibitor herbicids such as Chlorsulfuron, imazethapyr, flumetsulam and penoxsulam.The characteristics of Acetohydroxyacid synthase were determined. Vmax is 0.568 mmol/h, while Km is 18.38 mM according to the kinetic equation. Lm10’s AHAS showed resistance to high concentration of metsulfuron-methyl. The activity of enzyme was still up to 50% when the concentration of metsulfuron-methyl reached 2500μmol/L . The the optimum pH is 6.5, and the optimum temperature is 30℃. Acetohydroxyacid synthase showes feedback regulatory to the end products Val. It don’t showes its sensitivity to the organic solvent. Five metal ions were chosen to study their effects on the Acetohydroxyacid synthase activity, and the results showed that when the metal ions concentration reached 2 mmol L^-1, Fe²⁺、Ca²⁺could enhance the enzyme activity.The acetohydroxyacid synthase genes z7vIH was amplified from the genome DNA of strains Lm10. The entire nucleotide sequences of ilvI, ilvH were 1725bp, 492bp in length, which encoded two polypeptides of 575, 164 amino acid residues respectively. The ilvI and ilvH were cloned into the bacterial expression vector pET29a（+） respectively, and functional expressed in the Escherichia coli strain BL21（DE3）. Howere the regulatory subunits did not functional expressed in the Escherichia coli when the ilvIH cloned into expression vector pET29a（+）. It may be not recognized by the Escherichia coli’s translation system. The expressed production pET-I displayed acetohydroxyacid synthase activity and showed resistance to high concentration of metsulfuron-methyl. The ilvIH were highly conserved in Pseudomonas sp ,and The alignment result of the ilvIH amino acid sequence showed that ilvI of strains Lm10 differed from that of strain KT2440 by 6 sites,which are N134H、P135A、T136S、V210I、Y214F and W486S. While the ilvH of the two strains were the same. The roles of these 6 different sites from that of strain Lm10 will be determined by using site-directed mutagenesis. Resistant mutations of this enzyme have theoretical value and potential application in plant transgenetic engineering.