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[1]何 强 杨昭杰 季秀玲 魏云林 林连兵** 张 琦**.一个硫化叶菌病毒启动子的分离与鉴定*[J].应用与环境生物学报,2019,25(02):1-12.[doi:10.19675/j.cnki.1006-687x.2018.06015]
 HE Qiang,YANG Zhaojie,JI Xiuling,et al.Isolation and identification of a Sulfolobus virus promoter*[J].Chinese Journal of Applied & Environmental Biology,2019,25(02):1-12.[doi:10.19675/j.cnki.1006-687x.2018.06015]
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一个硫化叶菌病毒启动子的分离与鉴定*()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
25卷
期数:
2019年02期
页码:
1-12
栏目:
研究简报
出版日期:
2019-04-25

文章信息/Info

Title:
Isolation and identification of a Sulfolobus virus promoter*
文章编号:
201806015
作者:
何 强 杨昭杰 季秀玲 魏云林 林连兵** 张 琦**
昆明理工大学生物工程技术研究中心 昆明 650500
Author(s):
HE QiangYANG ZhaojieJI XiulingWEI YunlinLIN Lianbing**&ZHANG Qi**
Kunming University of Science and Technology Bioengineering Technology Research Center, Kunming 650500,China
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
Keywords:
STSV2 virus Promoter screening plasmid Promoter P37 Promoter activity Core promoter sequence element subsequence
DOI:
10.19675/j.cnki.1006-687x.2018.06015
摘要:
很多研究已表明,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。为了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,本研究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。这些结果表明,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)
Abstract:
Many studies have shown that there are no homologous protein-encoding sequences of eukaryotic basal transcriptional machinery such as RNA polymerase (RNAP) in the genomes of almost all archaeal viruses, and promoter activity may be important for the transcription of viral genes during infection.In order to further reveals the relationship between the sequence structure characteristics and the activity of archaeal viral gene promoters,the study f irst subcloned the β- galactosidase encoding gene lacS into 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 Sulfolobus islandicus strain E233S for function al analysis. The result showed that the lacS gene was successfully expressed in 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 bp sequence upstream of capsid-encoding gene ORF37 of virus STSV2 , and the promoter activity was further analyzed by transforming pSeSD-P37-lacS into E233S strain. The results of 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, that indicated the P37 was a functional promoter fragment and exhibit ed almost equiv alent activity with the araS promoter. Further sequence analysis also showed that P37 contained bas al promoter sequence elements of Sulfolobus genes, such as initiator, TATA-box and BRE.?These results indicated 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.

备注/Memo

备注/Memo:
收稿日期: 2018-06-08 接受日期 Accepted: 2018-07-20
*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(KKSA201126005)和教育部回国人员科研启动基金(KKQA201226003)资助?
**通讯作者(E-mail: linlb@sohu.com;qzhang37@ gmail.com)
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更新日期/Last Update: 2018-08-02