Construction Of Molecular Screening Model Of Acetohydroxyacid Synthase In Vitro And Interaction With Herbicide

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families.The innovations of this program were the expression of plant AHAS target protein which exists in Arabidopsis thaliana and construction of screening model in vitro, which constructed prokaryotic and eukaryotic expression system. The activity of expressed production and the activity variety of the interaction between expressed production and herbicide were mensurateted and study on the interaction between AHAS and chlorimuron-ethyl, Imazethapyr and monosulfuron intermediates by Fluorescence Spectrometry. And the results were as follows:(1). Extraction of Arabidopsis thaliana total RNA, and according to AHAS catalytic subunit gene sequence from NCBI gene bank (sequence NO. NM 114714), sense and anti-sense primers were designed and synthesized. And the target gene was amplified by RT-PCR. The result of sequencing showed that the AHAS catalytic subunit gene's CDS includes 2013 nt and codes 670 amino acid. The sequence result was submited to NCBI gene bank and obtained the sign NO.DQ991161. The homology of nucleotides is 99% compared with the reported one (sequence NO. NM 114714).(2). According to multiple cloning sites of prokaryotic expression plasmid pET-28a(+) and restriction map of the cloned AHAS catalytic subunit gene, another pair of primers were designed and synthesized, then the gene including restriction sites of Not I and Sal I was amplified and ligased with plasmid pET-28a. Through restriction digesting and PCR identification, it suggested that recombined expressing vector was successfully constructed. And the vector was transformed into E.coli BL21 and induced to express with IPTG. The result of SDS-PAGE analysis of expressed production show that AHAS had been expressed, but informed including body which is about 69 KDa.(3). According to the multiple cloning sites of eukaryotic expression plasmid pPIC9K and restriction map of the cloned AHAS catalytic subunit gene pMD19-T-AHAS, one pair of primers were designed and synthesized, then the gene including restriction sites of Not I was amplified by PCR and ligased with plasmid pPIC9K. Through restriction digesting and PCR identification, it suggested that recombined expression vector was successfully constructed. And the vector was transformed into pichia pastoris GS115, recombination Mut⁺ was screened and induced to express with methanol. The result of SDS-PAGE analysis of expressed introduction identification suggested that we obtained recombined expression protein, which is about 72.5 KDa can secrete to culture medium.(4). Purifiction expressed protein in E.coli under denaturing condition by Ni-NTA superflow cartridger, denatured it by dialyse and identificated its activity.Activity is 1.18×10^-8μmol/min and specific activity is 1.31μmol /mg·min. Activity is 1.11×10^-8μmol/min and specific activity is 0.59μmol /mg·min.(5). The interaction of chlorimuron-ethyl (CE), Imazethapyr (IQ), monosulfuron intermediates and acetohydroxyacid synthase (AHAS) under solution condition was studied by fluorospectrophotometry. The experiment demonstrated that the quenching mechanism of CE, IQ and AHAS was static quenching process. The quenching constant is 4.73×10¹² and 1.16×10¹² L·moprokaryotic·s and the binding sites (n) were 1.0649 and 1.3813.According to the F(o|¨)rster nonradiative energy transfer theory, the binding distance between donor(AHAS)and acceptor (CE)was calculated to be 0.85 nm,acceptor(IQ) was 0.399 nm presuming that the interaction of CE, IQ and AHAS was driven mainly by hydropHobic force. The influence of the presence of CE on structure of AHAS was studied by synochronous fluorescence method, it is convenience to screen novel herbicide compound and identify molecular mechanism. Monosulfuron intermediates have no function with AHAS.