【作者】 冀伟；

【导师】 李子义；

【作者基本信息】 吉林大学 ， 动物学， 2013， 博士

【摘要】 甾体类化合物是自然界中广泛存在的一类天然化学成分，包括植物甾醇、C21甾类、昆虫变态激素、强心苷、胆汁酸、蟾毒配基、甾体生物碱、甾体皂苷等。虽然种类繁多，但它们的结构中都具有重要的相似结构——环戊烷骈多氢菲的甾体母核。随着环境污染的日益加重，由于甾体类化合物结构与人和动物的激素结构相似，影响人和动物的正常发育，使得健康受到威胁。睾丸酮丛毛单胞菌（Comonas testosteroni）是一种已经被证明的能够利用甾体类化合物作为唯一碳源并降解甾体类化合物的革兰氏阴性菌。本文通过对7α-羟基类固醇脱氢酶基因的克隆进行原核表达，将纯化的蛋白质制备抗体，通过酶联免疫吸附技术测定在有激素诱导情况下7α-羟基类固醇脱氢酶表达量明显提高。通过对7α-羟基类固醇脱氢酶敲除得到突变菌株，发现敲除7α-羟基类固醇脱氢酶后的Comonas testosteroni突变菌株无论在生长速度和甾体类化合物降解速度上就有明显降低。在其表达调控方面，通过对其上游LysR基因的质粒共转化在无激素诱导情况下7α-羟基类固醇脱氢酶表达量较低，在有激素存在条件下表达量提高。初步认为LysR对于7α-羟基类固醇脱氢酶为负调控。更多还原

【Abstract】 With the development of industrialization, more and more environmentalpollutants are discharged into the nature, including organic pollutants. Due to thesimilar structure with human and animal hormone, the increasing concentration ofsteroidal compounds affects the growth and development of human beings andanimals,which causes the dysplasia of animals and the metabolic diseases of humanbeings. The traditional technology in dealing with the pollutants has the short-comings of low efficiency, high-cost, and easily causing the second pollution by thetoxic and hazardous intermediates. Nowadays the bioremediation degradation ofsteroidal compounds is a quite hot research topic in solving the environmentalpollution.Comamonas testosteroni is a bacterium that can use steroidal compounds assole carbon source for growth and metabolism of Gram-negative bacteria.Comamonas testosterone bacteria metabolic pathways of degradation steroidalcompounds are complex, about dozens of kinds of enzymes involved. Among themany enzymes7α-hydroxysteroid dehydrogenase capable of specifically acting onthe steroid nucleus7carbon atoms, hydroxyl group α to oxidation. Comamonastestosterone ATCC11996sequencing work has been completed, found in itsgenome does exist another7α-hydroxy steroid dehydrogenase possible sequences.Get the PCR production of7α-hydroxysteroid dehydrogenase gene and itsneighboring LysR, and to analyze the conserved sequences, presumably its basiccharacters. After7α-hydroxysteroid dehydrogenase gene and its neighboring LysR get purified protein expression vector. And get7α-hydroxysteroid dehydrogenasepolyclonal antibodies, established ELISA methods. ELISA method proved7α-hydroxysteroid dehydrogenase expression can be changed by vary steroidhormone changes.The7α-hydroxysteroid dehydrogenase gene and its300bp regulatorysequences upstream made plasmids were transformed into E. coli genes with LysRprove LysR gene is indeed involved in7α-hydroxysteroid dehydrogenase gene isregulated, when the presence of hormones7α-hydroxysteroid dehydrogenaseexpression was significantly increased. To further prove7α-hydroxysteroiddehydrogenase in the degradation of steroid compounds play an important role inthis study, suicide plasmid approach would make7α-hydroxysteroid dehydrogenaseknockout, and found that the mutation greatly reduced capacity in plant growth, anddegradation of hormones. Due to microbial degradation of pollutants is by variousenzymes, so the mode of enzymes and the regulation of its own way has a veryimportant significance. This research attempts7α-hydroxysteroid dehydrogenaseresearch, in order to further understand Comamonas testosterone mechanism ofaction of bacteria lay a foundation.更多还原