【作者】 何强；

【导师】 张琦；

【作者基本信息】 昆明理工大学 ， 生物化工， 2019， 硕士

【摘要】 病毒在感染宿主过程中通过编码特殊的转录调节蛋白直接结合DNA或竞争性结合宿主RNAP来调节宿主和自身基因的转录,并起始病毒相关的生命过程。虽然对细菌噬菌体和古细菌病毒的研究已初步揭示一些感染过程中病毒（或噬菌体）基因转录的调节机制,但古细菌病毒在感染过程中如何调节自身基因转录的研究鲜有报道。此外,目前所有报道的古细菌病毒转录调节因子都是以阻遏物的形式参与基因转录调节,而以转录激活因子的方式调节转录的转录调节蛋白鲜有报道。前期的研究表明,过表达硫化叶菌（Sulfolobus）病毒STSV2（Sulfolobus tengchongensis spindle-shaped virus 2）的转录调节因子编码基因ORF2可在病毒感染过程中显著提高宿主和病毒基因的mRNA转录水平,表现出普遍性转录激活因子的特点,但具体的作用机制仍不清楚。因此,本研究拟以硫化叶菌病毒STSV2编码的ORF2蛋白作为研究对象,对ORF2蛋白与病毒基因启动子的结合位点进行分析,同时对病毒STSV2衣壳蛋白基因ORF37的启动子结构和功能进行鉴定。ORF2蛋白是一种具有典型的winged HTH（wHTH）结构域特征的转录调节因子,为了分析ORF2蛋白在转录过程中与DNA的相互作用及具体的序列信息,首先PCR扩增ORF2基因,构建N端His标签标记的原核表达载体pET32aORF2,转化大肠杆菌表达宿主BL21（DE3）,诱导表达后使用Ni²⁺亲合纯化得到高纯度His标签标记的ORF2蛋白。同时分别扩增ORF2基因、ORF8基因和ORF37基因的上游500 bp潜在的启动子片段并将其进行生物素标记,采用电泳迁移率分析（EMSA）进行体外DNA与蛋白结合实验。结果显示,ORF2蛋白与这三个启动子片段都有相互作用,表明ORF2蛋白是一种普遍性的促进基因转录的转录调节因子。进一步将病毒衣壳蛋白编码基因ORF37的启动子片段P37-500片段分别截短基因上游216 bp和144 bp的两个片段P37-216和P37-144,通过EMSA分析两个片段与ORF2蛋白的相互作用,结果表明ORF2与两个短片段都有结合。为了进一步确定ORF2蛋白结合启动子DNA的序列信息,采用DNase I足迹实验分析了P2-500、P8-500和P37-500三个片段与ORF2蛋白结合的DNA结构,结果表明,ORF2蛋白与P2有两个结合位点47⁸1和130¹47,与P8有两个结合位点44⁹4和124¹47,与P37有三个结合位点46⁹4,118¹45和154¹75。分析所有的结合位点发现,每个结合位点的碱基数不一样,蛋白结合启动子时没有保守性很强的序列结构,但会对A/T有偏好;另外,最下游的结合位点包含了部分的BRE元件,提高了对RNAP复合物的招募促进基因转录。这与已报道的起转录阻遏作用的古细菌病毒含RHH结构域的转录调节结合方式不同。为了进一确定ORF2蛋白与DNA结合时具体的结构信息,对P37-500,P37-216和P37-144的启动子活性进行测定,确定其核心启动子区域的位置和其他启动子序列元件信息。首先我们在硫化叶菌表达载体pSeSD的基础上在araS启动子下游插入一个报告基因lacS,构建了一个古细菌病毒启动子筛选系统。利用P37-500,P37-216和P37-144启动子片段代替araS启动子,分别构建出启动子活性筛选载体pSeSDp37-500lacS、pSeSDp37-216lacS和pSeSDp37-144lacS,转化硫化叶菌E233S菌株,通过转基因菌株的β-半乳糖苷酶活性分析确定启动子序列结构信息和活性大小。分析结果显示,P37-144序列内包含了TATA-box、BRE（Transcription factor B Identification Element）、PPE（Proximal Promoter Element）和Inr（Initiator element）等核心启动子元件。启动子活性分析结果显示,三个P37启动子片段均为非阿拉伯糖诱导型启动子,且P37-500启动子活性与araS启动子的活性相似,但是P37-216和P37-144的启动子活性都远大于araS启动子,其中P37-144的活性最大为P37-500的2.1倍,P37-216的活性为P37-500的1.7倍。表明P37启动子-144位点上游可能存在某些阻遏元件影响基因的转录,但具体序列信息还需要进一步研究揭示。综上所述,硫化叶菌病毒STSV2转录调节蛋白ORF2能与多个病毒基因启动子相互作用,是一种普遍性的促进基因转录的转录调节因子,它结合DNA时没有较保守的序列结构,但会对A/T有偏好,同时下游的位点中包含了部分BRE位点,提高对RNAP复合物的招募,但具体的作用机制和相关的调控过程还需要深入研究。对P37片段进行核心启动子元件和活性分析,结果显示P37-144中包含了TATA-box、BRE、PPE和Inr核心启动子元件,且P37-144片段的启动子活性最高,P37-216片段次之,P37-500最低,证明了在P37-144上游可能有一些阻遏元件的存在。本研究有助于揭示ORF2促进基因转录的调节机制,为揭示STSV2病毒感染宿主时的转录过程和其他生命活动打下基础,同时对研究古细菌病毒的起源、进化和病毒与宿主的关系提高参考。更多还原

【Abstract】 The virus can modulates the transcription of the host and its own genes by virus-encoded specific transcriptional regulatory proteins directly binding to the DNA or competitively binding to the host RNAP during infection,and initiates a virus-related life process.Although studies of bacteriophages and archaeal viruses have primarily revealed some regulatory mechanisms of viral（or phage）gene transcription during infection,but little is known about the regulation mechanisms of archaeal viral gene transcription during infection.In addition,all of the reported archaeal viral transcriptional regulators are functioned as repressors for the gene transcriptional regulation.So far,no viral regulatory proteins as transcriptional activator to regulate the gene transcription during infection was reported.Our previous studies have shown that ave shown that overexpression of the transcriptional regulator-encoded gene ORF2 of virus STSV2（Sulfolobus tengchongensis spindle-shaped virus 2）in Sulfolobus islandicus E233S can significantly increase the mRNA transcription levels of host and viral genes during viral infection,indicating the characteristics of a gobal transcriptional activator.However,the specific regulation mechanisms of ORF2 remains unclear.Therefore,the virus STSV2-encoded ORF2 protein was used in this study to analyze the binding sites of viral gene promoters,and analysis of the structure characteristics and function of STSV2 capsid protein gene ORF37 were simultaneously carried out.ORF2 protein is a transcriptional regulator with typical winged HTH（wHTH）domain.To analyze the interaction of ORF2 protein with DNA and specific sequence information of binding sites during transcription,the ORF2 gene was firstly amplified by PCR,which was further used to construct a recombinant prokaryotic expression plasmid pET32aORF2 by fusing his tag to the N-terminal of ORF2 protein.The resultant plasmid was further transformed into E.coli BL21（DE3）for expression.After inducing expression,the his-tagged ORF2 protein was purified by Ni²⁺affinity chromatograph.At the same time,the upstream⁵00bp promoter fragments of ORF2gene,ORF8 gene and ORF37 gene were amplified and labeled with biotin,respectively.Electrophoretic mobility shift assay（EMSA）was performed to analyze in vitro the interaction of ORF2 protein with these three promoters.The results showed that the ORF2 protein interacted with these three promoter fragments,indicating that the ORF2 protein is a global transcriptional regulator that promotes gene transcription.Further,based on the promoter fragment P37-500,two shorter fragments P37-216 and P37-144,upstream of the viral capsid protein-coding gene ORF37,were obtained for same EMSA analysis.The results showed that ORF2 also could bind to both two short fragments.In order to further determine the sequence information of ORF2 protein-binding promoter DNA,the DNA structure of the two fragments of P2-500,P8-500 and P37-500 bound to ORF2 protein was analyzed by DNase I footprint assay.The results showed that ORF2 protein and P2 have The two binding sites 47-81 and 130-147 have two binding sites 44-94 and 124-147 with P8,and three binding sites 46-94,118-145and 154-175 with P37.Analysis of all binding sites revealed that the number of bases per binding site was different,and the protein-binding promoter did not have a highly conserved sequence structure,but would have a preference for A/T;in addition,the most downstream binding site The inclusion of a portion of the BRE element enhances the recruitment of RNAP complexes to facilitate gene transcription.This is in contrast to the reported transcriptional regulation of the RHH domain of the archaeal virus that acts as a transcriptional repressor.To further determine the specific structural information of the ORF2protein-bound promoter,the promoter activities of P37-500,P37-216 and P37-144were analyze to identify the core promoter region and other elements.First,a reporter gene lacS was inserted at downstream of the araS promoter,based on the Sulfolobus expression vector pSeSD,to construct an archaeal viral promoter screening system.The three promoter fragments P37-500,P37-216 and P37-144 were used to replace the native araS promoter to constructed the promoter activity screening vectors pSeSDp37-500lacS,pSeSDp37-216lacS and pSeSDp37-144lacS,respectively,and further transformed the E233S strain.The structural information of the promoters and their activity were determined by theβ-galactosidase activity of the transgenic strains.The analysis results showed that the P37-144 sequence contained core promoter elements such as TATA-box,BRE（Transcription factor B Identification Element）,PPE（Proximal Promoter Element）and Inr（Initiator element）.Promoter activity analysis showed that the three P37 promoter fragments were not inducible promoters,and the P37-500 promoter exhibited similar activity to that of the araS promoter,but the promoter activities of P37-216 and P37-144 were higher than that of the araS promoter.The activity of P37-144 is 2.1 times higher than that of P37-500,and the activity of P37-216 is 1.7 times higher than that of P37-500.These results indicated that some repressor elements might localized at the upstream of the-144bp site of the P37 promoter,but the specific sequence information is still needed to be further revealed.In summary,the S.oxysporum STSV2 transcriptional regulatory protein ORF2interacts with multiple viral gene promoters and is a universal transcriptional regulator that promotes gene transcription.It binds DNA without a more conserved sequence structure.However,there will be a preference for A/T,and some downstream BRE sites will be included in the downstream site to enhance the recruitment of RNAP complexes.However,the specific mechanism of action and related regulatory processes need further study.The core promoter element and activity analysis of the P37 fragment showed that the T37-144 contained the TATA-box,BRE,PPE and Inr core promoter elements,and the P37-144 fragment had the highest promoter activity and the P37-216 fragment.The lowest P37-500 proves that there may be some repressive elements upstream of P37-144.This study will help to reveal the regulatory mechanism of ORF2 to promote gene transcription,to lay the foundation for revealing the transcription process and other life activities of STSV2virus infecting host,and to improve the research on the origin and evolution of archaeal virus and the relationship between virus and host..更多还原