Trichlorophenol Synthesis And Degradation Of Functionally Related Genes In Gene Analysis -PsUGT2I

Trichlorophenol, containing6types of isomers, is a kind of chlorinated aromatic ring compounds which is hard to degrade in natural environment. It also has the effects such as carcinogenic, teratogenic and mutagenic effects to human physical health. The previous chemical-and physical-degradation technologies are usually costly, inefficient, or even caused secondary pollution. Therefore, a safe, fast, efficient, and economical novel remediation approach is an urgent need. In recent years, a number of studies have focused on transgenic plant to biodegrade these highly toxic aromatic compounds. This biodegradation combining plant and microbe enhances the degradation of or the conversion of aromatic compounds.Glycosyltransferase, belonging to a superfamily, catalyze glycosidic bond formation using activated donor sugar substrate in vivo. The acceptors are different, such as protein, nucleides, oligosaccharides, fat, small molecular etc. The products are always polysaccharides, glycoprotein, glycolipid and so on. In plants, glycosyltransferases mostly are involved in glycosylation which is an important step of the secondary metabolites. It has been reported that this process was related with the transformation and degradation of aromatic hydrocarbons and chlorophenols. In this research, the gene (PsUGT2I) of UGT2from Picea sitchensis published in EMBL database was chemically synthesized according to plant codon preference via the PCR-based two-step DNA synthesis (PTDS) method. The amino acid sequence of synthesized PsUGT2I was identical with the amino acid sequence of interest.In order to analyze the function of PSUGT2I, we construct binary vector, electroporate into Agrobacterium, use the floral dip method to do the transformation of Arabidopsis thaliana. After screening with hygromycin medium, extracting RNA of the plant to PCR, the positive transgenic strain was found. Physiological analysis of transgenic plants showed that, relate to wide type, they perform a better tolerance of2,4,5-TCP and lower content of soluble sugar. The results of HPLC analysis further confirmed that the protein extracts of positive plants also possess more ability to degrade of trichlorophenol than wild type. We also comfirmed that recombinant yeast (pichia pastoris) containing this gene exhibited a better tolerance on the selective media with2,4,5-TCP. The deduced molecular mass of recombinant PsUGT2I was extracted by liguid nitrogen grinding method and was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(54kDa). HPLC method showed that the recombinant yeast can reach the degradation of43.3%2,4,5-TCP at one hour in vitro. These results will provide a novel approach for the phytoremediation and microbial remediation of trichlorohpenol.