tac启动子碱基突变对睾丸酮丛毛单胞菌(Comamonas testosteroni)降解能力的影响  

Influence of Mutant tac Promoter on the Degradation of Comamonas testosteroni

作　　者：陈建秋[1] 潘大仁[1] 周以飞[1] 程晓春[2] 张剑亮[1] 卢文标[2] 陈雅艳[1] 林钿[1] 

机构地区：[1]福建农林大学作物科学学院,福州350002 [2]福建农林大学生命科学学院,福州350002

出　　处：《农业生物技术学报》2008年第2期336-340,共5页Journal of Agricultural Biotechnology

基　　金：福建省科技厅攻关计划重大项目(No.2003N028);教育部科学技术重点项目(No.207056);福建省教育厅科学技术重点项目(No.JA06011);福建省发改委项目(闽计投资〔2003170号〕)资助

摘　　要：应用PCR技术将发生突变的tac启动子-10区(TATGTT)回复突变为TATATT和TATGAT,构建了2个重组质粒pPM1和pPM2。酶切结果发现,pPM1质粒转化大肠杆菌(Escherichia coli)后部分细菌对tac启动子进行切除。将重组质粒分别与pAX1共转化大肠杆菌,提取细菌总蛋白,酶联免疫吸附法测定细菌总蛋白中activator及3α-HSD的表达量,结果表明,tac启动子-10保守区为TATATT的质粒比-10保守区为TATGAT的质粒具有更高的转录活性,activator及3α-HSD的表达量高低依次均为pPM1>pPM2>pb(对照)。根据同源重组原理,将重组质粒电击转化睾丸酮丛毛单胞菌(Comamons testosteroni,C.T.),并利用HPLC测定了各突变菌对睾凡酮的降解能力,结果表明,不同突变菌对睾丸酮的降解能力高低依次为Cp7(C.T.+pPM1)>Cp4(C.T.+pPM2)>Cb7(C.T.+pb)>C.T.。-10 consensus sequence （TATGTT） of mutational tac promoter was reverted to TATATT and TATGAT by PCR amplification method. According to the results of PCR amplification, two recombinant plasmids pPM1 and pPM2 were constructed. Results of endonuclease digestion showed that tac promoter of plasmid pPM1 could be deleted in Escherichia coli. Total cell lysate of the host bacteria was extracted after co-transforming into E. coli HB101 respectively with pPM1＋ pAX1 and pPM2＋ pAX1. The quantity of activator or 3α- HSD protein was tested by ELISA. The results indicated that plasmid containing TATATT of -10 consensus sequence had higher transcriptional activity than that containing TATGAT, and the quantity of activator or 3α- HSD protein expressed in different plasmids was pPM1〉pPM2〉pb（control）. Meanwhile, recombinant plasmids were integrated into Comamonas testosteroni （C.T.）chromosome through homologous recombination by electroporation respectively. Their testosterone degradation ability was determined by HPLC and their testosterone degradation ability was Cp7（C.T. ＋ pPM1）〉Cp4（C.T. ＋ pPM2）〉 Cb7（C.T. ＋ pb）〉C.T..

关 键 词：tac启动子 突变 睾丸酮丛毛单胞菌 

分 类 号：S188[农业科学—农业基础科学]