Analysis Of Expressional Regulation Of3β/17β-hydroxysteroid Dehydrogenase Gene In Comamonas Testosteroni

Steroid hormones is the presence of an important class of pollutants in the environment and the type of steroid hormone biological degradation and degradation of the regulation of gene expression analysis increasingly affected by the attention of scientists. Comamonas testosteroni is a used of steroids as carbon source and energy of gram negative bacteria.In this paper, using Enzyme-linked immunosorbnent assay, gene knock-out, high performance liquid chromatography, circle of RT-PCR, Reverse PCR, Co-transformation of plasmids technology of Comamonas testosteroni in3β/17β-hydroxysteroid dehydrogenase induced expression, function analysis, as well as the transcription start site and expression regulation mechanism.In the culture medium were added to the equivalent of17β-estradiol, estriol, cholesterol, testosterone by Comamonas testosteroni cultured, ELISA results reveal3β/17β-HSD induced by steroid hormones, particularly testosterone on3β/17β-HSD induced effect, higher than that without steroid body hormone added45%of the expressed protein.The first construction of3β/17β-HSD gene deletion mutants study found:3β/17β-HSD on cholesterol, testosterone use play an important role.3β/17β-HSD gene deletion mutant strains for degradation of cholesterol by loss of function, on testosterone use efficiency decreased more than95%; at the same time found3β/17β-HSD the steroid hormone action target point is on C3carbonyl.By using the circle of RT-PCR method found3(β/17β-HSD mRNA of full-length is not unique, but gene transcription start sites focus on the basic gene upstream-143bp position, may be associated with bacterial metabolism and internal mechanism.The LysR transcriptional regulatory family is a class of transcription regulation factor. Using co-transformation of plasmid study of the gene upstream of an unknown protein LysR for3β/17β-HSD transcription expression and regulation studies found:in the absence of exogenous hormones on induction conditions, containing the LysR gene of strain ratio does not contain LysR gene of strain3β/17β-HSD expression increased24%; at the same time containing the LysR gene of strains of3β/17β-HSD expression quantity with steroidal hormone types vary, but were lower than those without hormone added expression, analysis of the LysR protein3β/17β-HSD plays the role of positive regulation.