The Research Of Different Herbicides' Modes Of Action And Modelling Of ACCase CT Domain

Plants, like all other living organisms possess a wide variety of lipid molecules each ideally suited to their location and role. In plants lipids have a special major function, as constituents of the extracellular surface layers, providing a protective barrier between the plant tissues and the external environments. Fatty acids are carboxylic acids with a hydrocarbon chain. Depending on the lipid source, this chain can vary in length, but is most commonly 16or 18 carbon atoms long.The fatty acid biosynthesis pathway is a primary metabolic pathway, because it is found in every cell of the plant and is essential to growth. Inhibitors of fatty acid biosynthesis are lethal to cells.The synthesis of fatty acids comprises two main events: the de novo biosynthesis of fatty acids, followed by further modification steps. De novo biosynthesis involves the combined activity of the two enzyme systems acetyl-coA carboxylase(ACCase) and fatty acid synthase(FAS). These are mainly plastidial events and in most plants, lead to the formation of the 16-carbon palmitic acid and the 18-carbon stearic acid, utilizing malonyl-coA as the source of condensing C2 unit. The long chain fatty acids can be elongated to very long chain fatty acids (VLCFAs) outside the plastid.Various herbicides which affect the lipid metabolism have been used commercially for many years. According to their target sites, modes of action, similarity of induced symptoms or chemical classes, the herbicides are classified into different groups by the Herbicide Resistance Action Committee (HRAC). For example, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs) are known as inhibitors of acetyl-CoA carboxylase in monocotyledon chloroplasts. In addition, pinoxaden, a known inhibitor of plastidic ACCase (IC50 0.1μM versus maize plastid ACCase) which also quite strongly inhibits cytosolic ACCase (IC50 17μM) was also studied. So it is maybe classified in"A"herbicides. acetamides (napropamide , diphenamid), chloroacetamides (dimethachlor) and carboxyamide (cafenstrol) belong to"k3"herbicides (HRAC classified) which are described by HRAC as inhibitors of cell division and VLCFAs synthesis in plants .According to the work which we did before, we disagree with the classic of the"k3"herbicides by HRAC. The effect of"k3"herbicides on the distribution of radioactivity between fatty acids synthesized from [1-¹⁴C] acetate/ [2-¹⁴C] malonate by different plants leaves, napropamide and diphenamid have no effect on VLCFAs. Therefore, they do not belong to"k3"herbicides. Dimethachlor and Cafenstrol inhibit the VLCFAs of barley and cucumber leaves, which are worthy of"k3"herbicides.Acetyl-CoA carboxylase (ACCase EC 6.4.1.2) plays critical roles in fatty acid metabolism in most organisms. It is a biotin-dependent enzyme that catalyzes the first committed step of de novo fatty acid biosynthesis. Evidences have indicated that, Gramineae ACCase in plastid is the target of two classes of herbicides, aryloxyphenoxypropionates (APPs) and cyclohexanediones (CHDs). These herbicides selectively inhibit the CT activity of ACCases and block the growth of gramineae grasses. However, the molecular recognition mechanism between CT domain of ACCase and inhibitors is not known. To systemically investigate these problems, it is necessary to get purified CT domain protein of ACCase.ACCase is the target of many herbicides, and the gene of yeast ACCase CT domain shared about 50% identity with plant. So we got the gene of ACCase CT from saccharomyces cerevisiae and expressed the bioactive enzyme in E.coli. This is a valuable modeling for the ACCase CT domain from other organisms.In this study, the plastid ACCase CT cDNA from saccharomyces cerevisiae was synthesized through reverse transcription (RT). Then the target gene was amplified by gene specific primers. Then the total CT domain gene was cloned into PET28a+ vector and transformed into E. coli. Hence we constructed the recombinant plasmid of CT domain gene, named YCT-his6. The recombinant plasmid was transformed into E. coli. and expressed by the induction of IPTG. BL21 (DE3) pLys (BLP) was selected as expression host and the expression conditions were optimized, including induced density of cells, concentration of IPTG, temperature and time and so on. The SDS-PAGE showed that YCT-his6 could be expressed in E. coli. and unfortunately, the recombinant protein was was over expressed in E. coli. as inclusion bodies. In order to recovering the soluble protein, we select a shorter gene which was cut 100 bp from saccharomyces cerevisiae ACCase CT domain. But the result of expression is the same as the whole long saccharomyces cerevisiae ACCase CT domain. So we decided to refold the YCT-his6 inclusion bodies.First, we research the washing, dissolving and refolding of the inclusion bodies. About 80% recombined protein in inclusion bodies after washing by low concentration of detergent and denaturant. Compared with different pH buffer, 8M urea in pH8.0 Tris is the perfect dissolving buffer for the inclusion bodies. Finally, we recovered about 90% denatured YCT-his6 by washing and purifying inclusion bodies.Second, we research about classify of herbicides using the 14C labeling experiment. Acetamides have no inhibition on the VLCFAs, we considered that acetamides are not belong to"k3"herbicides. Pinoxaden as a new herbicide inhibit de novo fatty acid synthesis of gramineous plant, it is the inhibitor of ACCase. We suggest that Pinoxaden should be belong to the"A"herbicides as"APPs"and"CHDs".So we got the gene of ACCase CT from saccharomyces cerevisiae and expressed the enzyme in E.coli as inclusion. The bioactive enzyme was recovered from refolding the inclusion. This is a valuable modelling for the ACCase from other organisms. It will be usful information for the mode of action research of new herbicides.