【作者】 戴艺民；

【导师】 潘大仁；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2005， 博士

【摘要】 人类活动将类固醇类物质(如雌二醇(E2)、口服避孕药)、多环芳香烃(PAHs)及杀虫剂等化合物大量排入环境中,这些物质已经引起生态破坏,不育、癌症等各种疾病日益增多。在众多的环境激素物质中,类固醇类物质不但非常稳定而且其激素活性大大高于其他环境激素类物质。C.testosteroni具有降解甾核的关键酶3α-羟类固醇脱氢酶/碳酰基还原酶(3α-hydroxysteroid dehydrogenase/carbonyl reductase,3α-HSD/CR)基因,因此能够以睾丸酮、孕酮和胆汁酸等类固醇类物质为唯一碳源和能源,通过包含许多酶的氧化甾核的复杂代谢途径,完全消化这类底物,因此该菌在这些危害大的稳定化合物的生物修复中起重要作用。然而,涉及这些物质降解的酶在该菌中是诱导表达的。为更好发挥C.testosteroni的作用,本研究对该细菌的一些基因进行克隆、载体构建和在大肠杆菌里进行表达:并对原始菌进行改造,检测。结果如下: 1.提取C.testosteron.的基因组DNA,利用PCR扩增3α-HSD/CR和activator基因.构建了相应的表达载体pET-15b-3α和pET-15b-actl,并在大肠杆菌BL21里进行了诱导表达,为蛋白质纯化、抗体的制备打下基础。 2.提取C.testosteroni的基因组DNA,利用PCR扩增activator基因。以具有tac强启动子的质粒pKtac1为载体,构建了2个含有C.testosteroni activator基因的质粒:pKtac1-actl(564bp全长activator基因)和pKtac1-act2(409bp部分activator基因)。这2个质粒经测序后发现:pKtac1-act1和pKtac1-act2在tac启动子的-35区保守序列都为TTGACA;在-10区保守序列pKtac1-actl为TATAAT,而pKtac1-act2却突变为TATGTT。将pKtac1-actl和pKtac1-act2分别进行酶切和连接,获得重组质粒pKtac1-act3(含启动子-10保守区为TATAAT,409bp的部分activator基因)和pKtac1-act4(含启动子-10保守区为TATGTT,564bp的全长activator基因)。 3.酶联免疫分析(ELISA)测定4个重组质粒分别转化(或与含3α-HSD/CR基因的质粒pAX1共转化)宿主菌Escherichia coli HB101的细菌总蛋白质中的activator和3α-HSD/CR的表达量。结果说明:启动子-10保守区为TATAAT的质粒比-10保守区为TATGTT的质粒有更高的转录活性;actvitor的过量表达可能影响质粒pKtac1-act3表达的稳定性;启动子-10区保守序列的下降突变(pKtac1-act2,TATAAT→TATGTT)及tac启动子的丢失(pKtac1-act3,继代培养4次)揭示了宿主菌E. coli HB101的自身防护机制;过高的细菌C.testosteroni activator表达可能对大肠杆菌有毒性,因此细菌启动本身的防护机制,对启动子保守区进行了下降突变修饰或者对强启动子进行剔除。 4.利用电穿孔转化法分别将质粒pKtac1-actl和pKtac1-act2同源整合到C.testosteroni的更多还原

【Abstract】 Chemicals such as steroids (e.g estradiol (E2) and oral contraceptive etc.) , polycyclic aromatic hydrocarbons (PAHs) and insecticide have been discharged into environment, which have already caused ecological destruction and various diseases such as infertility, cancer increased. Among these environmental hormones, steroid is quite stable and its hormonal activity much more harmful than other compounds in the environment. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) from Comamonas testosteroni is a key enzyme involved in the degradation of steroids and xenobiotic. C. testosteroni strains are able to use steroids (testosterone, progesterone, bile acid) as sole carbon and energy sources. Complete assimilation of steroids is achieved through a complex pathway involving many enzymatic steps of oxidation responsible for the breakdown of the steroid nucleus, C. testosteroni therefore play a significant role in the bioremediation of these stablely hormonally active compounds in the environment. However the catabolic enzymes for these compounds metabolism in C. testosteroni are not constitutively expressed but are induced by their respective substrates. For empowering C. testosteroni to degrade those compounds more effectively, we cloned 3α-HSD/CR and its activator genes from C. testosteroni. Then, its vectors were constructed and had been expressed in Escherichia coli. We constructed two genetically engineered bacteria of Comamonas testosteroni. The results were as follows:1. 3α-HSD/CR and its activator genes were cloned from C. testosteroni by PCR with Comamonas testosteroni genomic DNA. Then two expression vector pET-15b-3α and pET-15b-act1 were constructed respectively and expressed in E. coli BL21. All the above provided the foundation for protein purifing and antibody preparing.2. The activator gene from Comamonas testosteroni was amplied by PCR with Comamonas testosteroni genomic DNA. The PCR fragments were cloned to plasmid pKtacl (containing tac promoter) and two plasmids: pKtac1-act1 (containing 564bp total activator gene) and pKtac1 -act2 (containing 409bp partial activator gene) were obtained. The recombinant plasmid pKtac1-act2 was found mutation on promoter -10 consensus sequence (TATAAT-+TATGTT) by DNA sequencing. The inserts of pKtacl-act1 and pKtacl -act2 were subcloned into pKtacl and yielded pKtacl -act3 (-10 consensus sequence is TATA AT, containing 409bp activator gene) , pKtacl-act4 (-10 consensus sequence is TATGTT, containing 564bp activator gene) .3. The four recombinants were transformed into Escherichia coli HB101. The results indicated: 1) plasmids with TATA AT -10 consensus sequence had higher promoter activity than that of plasmids with TATGTT -10 consensus sequence in E. coli; 2) overexpression of activator from C. testosteroni was toxic to cells so pKtacl-ac/J which contains 409bp activator gene is unstable in E. coli HB101; 3) -10 consensus sequence down mutation (pKtac 1-ac/2, TATAAT-+TATGTT) and tac promoter fragment disappeared(pKtacl -act3 after 4 inoculations) from the cells revealed the self-defence mechanism off. coli HB101.4. The pKtacl -actl (containing 564bp total activator gene) and pKtacl-ac/2 (containing 409bp partial activator gene) were integrated into C. testosteroni chromosome through homologous recombination by electroporation respectively. Genetically engineered bacteria C. testosteroni+pKtac\-actl and C. /es/as/erom+pKtacl-ac/2 were gained by antibiotic and PCR selection then detected by Southern hybridization. The total cell lysate of C. testosteroni^ pKtacl-ac/7 and C. /estas/erom+pKtacl-ac/2 were extracted to detect the quantity of 3a-HSD/CR using ELISA. The results indicated that two genetically engineered bacteria could constitutively expressed 3a-HSD/CR gene evidently without the presence of substrates such as testosterone.5. Two genetically engineered bacteria were inoculated for 50 days and their total cell lysate were extracted per five days to detect the quantity of 3a-HSD/CR and activator using ELISA. The results indicated: the expression of activator and 3a-HSD/CR are quite high and stable when C. testosteroni^ pKtacl-ac/7 inoculated in LB medium containing antibiotic, while both unstable when inoculated in LB medium without antibiotic; the same as C. /es/os/erowj+pKtacl-ac/2. C. tes/as/erom+pKtacl-ac/./ was more stable than C. /es/as/erom+pKtacl-ac/2 in LB medium without antibiotic. From the results we could preliminary conclude that the expression of 3o-HSD/CR was regulated by the activator gene.6. The RT-PCR results indicted: genetically engineered bacteria C. testosteroni+pKtac\-act] and C. testosteroni+pKtacl-act2 constitutively expressed 3a-HSD/CR which is a key enzyme involved in the degradation of steroids and xenobiotic carbonyl compounds; at the same time 4-hydroxy-2-oxovalerate aldolase and 2-hydroxypenta-2,4-dienoate hydratase also constitutively expressed. These enzyme are both founctioned in the catabolism of AHs and steroids.7. The preliminary results of two-dimensional gel electrophoresis showed: genetically更多还原