Preliminary Study On The Susceptibility Of Herbicide-targeted Acetolactate Synthase To Inhibitors And Its Molecular Mechanism

In this paper, the function mechanisms of acetolactate synthase(ALS) inhibitors to ALS had been carried out, using the methods of molecular biology, physiology and biochemistry.1. Selective inhibition of three inhibitors to the acetolactate synthase(ALS) activity of three plants was studied. The result of ALS activity determination in vitro test indicated that the ALS of japonica rice, barnyardgrass and rapeseed were more sensitive to bispyribac-sodium and KIH-6127 than that of indica rice, while the susceptibility of ALS to bensulfuron-methyl showed little differences among these plants. The result of ALS activity determination in vivo test showed that the inhibition of bispyribac-sodium and KIH-6127 to the ALS of barnyardgrass and rapeseed was strong, while the ALS of japonica rice could recover from the inhibition. Bensulfuron-methyl showed a strong inhibition to the ALS of rapeseed and no effect on the ALS of other plants. All of these three inhibitors have a weak inhibition to the ALS from indica rice. It was indicated that the different susceptibility of ALSs to some inhibitors was one of the reasons of different behave of different plant to these inhibitors in the field.2. In order to understand the difference of susceptibility in gene level, we extracted the total RNA from Japonica rice (Xioushui 11), then the single strand cDNA was synthesized. A fragment of 3' terminal 1266bp of ALS coding region was amplified by RT-PCR, and then was cloned into pGEX-T easy vector and sequenced. Ammo acid sequence comparison showed that the sequence shared 98.3% and 97.9% indentities with ALS genes from the sensitive and resistive Oryza saliva, respectively. The pariware indentities were from 90.1% to 91.7% between our sequence and the ALS genes from Zea mays, Hordeum vulgare, Triticum aestivum, Lolium multiflorum, Bromus tectorum, respectively. The indentitis were 71.7% and 72.9% with ALS genes from Nicotiana tabacum and Arabidopsis thaliana, respectively. The indentities were above 70% with ALS genes from other plants. The 585th and 664th amino acid in ALS were thought to concern to the susceptibility of Oryza saliva to ALS inhibitors.3. For getting pure ALS for inhibitor screening directly and studying further ALS function position, the partial ALS gene was inserted into donor plasmid pBacHTeGFP.The target gene was transposed to the target Bacmid in E.coli (DH10) by Tn7 transposition function. The recombinant Bacmid DNA was transfected into Tn-5B1-4 cells mediated by Lipofectin. The cytopathic effect (CPE) of the cells and detection of green fluorescence showed that the target fusion gene had already been expressed in Tn cell.