不同-10区保守序列的tac启动子表达睾丸酮丛毛单胞菌中的活化因子  

Expression of Activator from Comamonas testosteroni with tac Promoter Containing Different-10 Consensus Sequences

作　　者：戴艺民 [1] 周以飞 [2] Guangming Xiong  吴明霞 [2] 潘大仁 [2] 

机构地区：[1]福建农林大学作物学院,福州,350002,福建省农业科学院甘蔗研究所,漳州,363005 [2]福建农林大学作物学院,福州,350002 [3]Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School, Schleswig-Holstein, Campus Kiel,Brunswiker Str. 10, 24105 Kiel, Germany

出　　处：《农业生物技术学报》2005年第6期747-753,共7页Journal of Agricultural Biotechnology

基　　金：福建省科技厅攻关计划重大项目(No.2003N028);福建省发改委项目资助

摘　　要：提取睾丸酮丛毛单胞菌(Comamonastestosteroni)的基因组DNA,利用PCR扩增活化因子(activator)基因。将扩增产物克隆到pKtac1(含强启动子tac)中,构建含全长activator基因(564bp)的重组质粒pKtac1-act1和含部分activator基因(409bp)的pKtac1-act2。测序结果发现重组质粒pKtac1-act2的tac启动子-10保守区由TATAAT突变为TATGTT。将pKtac1-act1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)。将4个重组质粒分别转化入宿主菌Es-cherichiacoliHB101中,用酶联免疫分析方法测定细菌总蛋白质中的activator表达量;将pAX1(含3α-hsd/CR基因)分别和4个重组质粒一起转化入宿主菌E.coliHB101中,测定细菌总蛋白质中的3α-hsd/CR和activator的表达量。结果表明: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;过量的actvitor表达可能影响质粒pK-tac1-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(pK-tac1-act3,继代培养4次)揭示了宿主菌E.coliHB101的自身防护机制。The activator gene from Comamonas testosteroni was amplified by PCR with Comamonas testosteroni genomic DNA. The PCR fragments were cloned to plasmid pKtac1 （containing tac promoter） and two recombinant plasmids： pKtacl-act1 （containing 564 bp total activator gene） and pKtac1-act2 （containing 409 bp 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 pKtac1-act1 and pKtac1-act2 were subcloned into pKtacl and yielded pKtac1-act3（-10 consensus sequence is TATAAT, containing 409 bp activator gene）, pKtac1-act4 （- 10 consensus sequence is TATGTT, containing 564 bp activator gene）. The four recombinants were transformed into Escherichia coli HB101. The results indicated： 1） plasmids with TATAAT-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 pKtac 1-act3 which contains 409 bp activator gene is unstable in E. coli HB 101 ; 3） - 10 consensus sequence down mutation （pKtac1-act2, TATAAT→TATGTT） and tac promoter fragment disappeared（pKtac1-act3 after 4 inoculations） from the cells revealed the self-defence mechanism of E. coli HB101.

关 键 词：tac启动子 -10区保守序列 睾丸酮丛毛单胞菌 活化因子 基因表达 

分 类 号：Q781[生物学—分子生物学] X172[环境科学与工程—环境科学]