一个硫化叶菌病毒启动子的分离与鉴定  

Isolation and identification of a Sulfolobus virus promoter

作　　者：何强[1] 杨昭杰[1] 季秀玲[1] 魏云林[1] 林连兵[1] 张琦[1] HE Qiang;YANG Zhaojie;JI Xiuling;WEI Yunlin;LIN Lianbing;ZHANG Qi(Bioengineering Technology Research Center, Kunming University of Science and Technology, Kunming 650500, China)

机构地区：[1]昆明理工大学生物工程技术研究中心,昆明650500

出　　处：《应用与环境生物学报》2019年第2期399-404,共6页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(31260034;31660454);云南省应用基础研究基金资助项目(K KSA201126005);教育部回国人员科研启动基金(KKQA201226003)资助~~

摘　　要：几乎所有古菌病毒基因组中无RNA聚合酶(RNA polymerase,RNAP)等组成基本转录装置的同源蛋白编码序列,而且启动子活性对病毒感染过程中病毒基因的转录上可能具有重要的影响.为进一步揭示古菌病毒基因启动子的序列结构特点和活性之间的关系,首先基于硫化叶菌质粒pSeSD,将β-半乳糖苷酶编码基因lacS克隆到阿拉伯糖启动子araS下游多克隆位点,构建重组表达载体pSeSD-lacS.将pSeSD-lacS转化冰岛硫化叶菌(Sulfolobus islandicus)E233S菌株后的功能分析结果表明,lacS基因成功表达.在此基础上,利用硫化叶菌病毒STSV2衣壳蛋白编码基因ORF37上游500bp的潜在启动子片段P37替换pSeSD-lacS中的araS启动子,构建出新的重组表达质粒pSeSD-P37-lacS,进一步将pSeSD-P37-lacS转化E233S菌株进行启动子活性分析.β-半乳糖苷酶酶活结果显示,诱导后araS启动子酶活为14 345.7±422.3 mU,P37酶活为13 723.1±370.9 mU,表明P37片段具有启动子功能,而且活性与araS启动子相当.序列分析也显示,P37具有与硫化叶菌基因启动子类似的基础序列元件initiator、TATA-box及BRE等.本研究表明pSeSD-lacS可作为一个硫化叶菌病毒基因启动子筛选载体,而且高活性的基因启动子可能在STSV2病毒生命过程具有重要的作用.(图4表1参27)Many studies have shown that there are no homologous protein-encoding sequences of eukaryotic basal transcriptional machinery such as RNA polymerase in the genomes of almost all archaeal viruses, and promoter activity may be important for the transcription of viral genes during infection. To further determine the relationship between the sequence structure characteristics and the activity of archaeal viral gene promoters, the present study first subcloned the β-galactosidase encoding gene lacS into a multiple clone site downstream of the arabinose promoter araS on vector pSeSD to generate the recombinant expression plasmid pSeSD-lacS, which was further transformed into a Sulfolobus islandicus strain E233S for functional analysis. The results showed that the lacS gene was successfully expressed in the E233S strain.?Based on this, a new recombinant expression plasmid pSeSD-P37-lacS was constructed by replacing the araS promoter on the pSeSD-lacS with a potential promoter fragment P37, a 500-base pair sequence upstream of the capsid-encoding gene ORF37 of virus STSV2, and the promoter activity was further analyzed by transforming pSeSD-P37-lacS into the E233S strain.β-galactosidase activity showed that the araS promoter activity was 14 345.7 ± 422.3 mU after induction and the P37 activity was 13 723.1 ± 370.9 mU, which indicated that P37 was a functional promoter fragment and exhibited almost equivalent activity with that of the araS promoter.?Further sequence analysis showed that P37 contained basal promoter sequence elements of Sulfolobus genes, such as initiator, TATA-box, and BRE.?These results indicate that pSeSD-lacS can be used as a screening vector for the identification of promoters from Sulfolobus viruses and the high activity of viral gene promoters may play an important role in the life process of the virus STSV2.

关 键 词：STSV2病毒 启动子筛选质粒 启动子P37 启动子活性 核心启动序列元件子序列 

分 类 号：Q933[生物学—微生物学]