The Study On Stress Response Genes In RO4 During Metabolism Of Polychlorinated Biphenyls

Because of highly resistant to acid and alkali, oxidation resistance, heat resistance, polychlorinated biphenyls (PCBs) are often used for industrial production. With a lot of accumulation in adipose tissue, polychlorinated biphenyls (PCBs) cause serious harm to human body. Biodegradation was efficient, cheap and no secondary pollution, so get more and more people’s attention, and become the one of most perspective remediation of PCB. Rhodococcus sp. R04 is an efficient degradation strain of PCBs. Its transcriptome data indicated that the metabolite, stress response and some of membrane protein genes were up-regulated during culture of R04 with biphenyl.Research object for manganese catalase (RHOGL009077), regulating genes (RHOGL007659), and membrane protein (RHOGL009301). By the chemical synthesis and PCR bypass methods, the full length of Mn-CAT gene was obtained. Expression vectors of Mn-CAT and Mn-CAT-C gene were constructed, subsequently, introduced into E.coli BL21 (DE3). Mn-CAT-C was purified by Q-sepharose and ammonium sulphate precipitation. Mn-CAT was purified by Q-sepharose and Cobalt column. The enzymology properties was studied. A manganese catalase lacking n-terminal has been discovered. The lack of n-terminal affected the enzyme activity and the enzyme stability.The deficient vector was constructed and transformed into Rhodococcus sp. R04 by electroporation. Deficient strains were obtained by homologous recombination. Deficient strains could not metabolize of polychlorinated biphenyls after the knockout of regulating gene (RHOGL007659). By examining the Gene expression quantity of PCB degradation gene, including BphAl, BphB, BphC and BphD, it was found that the lack of RHOGL007659 affected degrading polychlorinated biphenyls. The co-expression vectors of membrane protein (RHOGL009301) and GFP was constructed, after exciting by 488nm fluorescence, the fluorescence was found around the cell, which proved the gene to be membrane protein. Compared with wild strains under the biphenyl culture conditions, deficient strains grow more slowly and delayed stable phase.High concentration of ROS produced by R04 during metabolism; of polychlorinated biphenyls. Manganese catalase (RHOGL009077) retained the majority of the active properties of the original enzyme, including ROS clearance. Lack of n-terminal leads to serious change in catalytic mechanism. RHOGL007659 may regulate the expression of PCB degradation gene other than that of BphD. It is presumed that membrane protein (RHOGL009301) may be involved in transporting the product in downstream pathways of polychlorinated biphenyls degradation.