家蚕核型多角体病毒lef5基因对病毒基因组复制和转录调控的影响  

The Effects of Bombyx mori Nucleopolyhedrovirus lef 5 Gene on Viral Genome Replication and Transcriptional Regulation

作　　者：余海鹏[1,2] 李俊[1,2] 张婷[1,2] 陈滨[1,2] 潘慧慧[1,2] 全滟平[1,2] 于威[1,2] 

机构地区：[1]浙江理工大学生物化学研究所,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2015年第5期895-901,共7页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2011AA100603);国家自然科学基金项目(No.31101831)

摘　　要：lef5是杆状病毒的核心基因之一。构建家蚕核型多角体病毒(BmNPV)lef5基因敲除型病毒lef5-ko-Bacmid和补回型病毒lef5-re-Bacmid,研究lef5基因在BmNPV的基因组DNA复制及各时期基因转录调控中的作用。透射电子显微镜下观察lef5-ko-Bacmid转染BmN细胞后不能产生具有侵染活性的病毒粒子(BV)。荧光定量PCR(qPCR)发现lef5基因缺失对病毒基因组DNA的复制没有明显影响,但会导致病毒各时期基因的转录水平显著下降。进一步通过检测由BmNPV多角体蛋白基因(polh)启动子驱动的荧光素酶活性变化,明确lef5基因对polh基因启动子的调控作用,结果表明lef5基因对polh基因启动子有显著的正调控作用。研究结果提示,lef5基因缺失后不影响BmNPV基因组DNA的复制,但会影响病毒早期、晚期、极晚期基因的转录表达。lef5 is one of the core genes of baculovirus. In order to study the roles of lef5 gene in Bombyx mori nucleopolyhedrovirus（ BmNPV） genomic DNA replication and gene transcriptional regulation at different stages,a lef5-knock out Bacmid（ lef5-ko-Bacmid） and lef5-repair Bacmid（ lef5-re-Bacmid） were prepared. After lef5-ko-Bacmid DNA was transfected into BmN cells,no infectious budded virus（ BV） was observed under transmission electron microscope. Real-time fluorescent quantitative PCR（ qPCR） showed that the absence of lef5 gene had no obvious effect on viral genome replication,but the gene transcriptional levels at different stages were reduced remarkably. In addition,in order to explore the regulatory function of lef5 gene on polyhedrin promoter,the activity of luciferase driven by polyhedrin promoter was detected.The results showed that lef5 gene had a positive regulatory effect on polyhedrin promoter. In summary,the deletion of lef5 gene did not affect the replication of BmNPV genomic DNA,but affected the transcription of viral early,late and very late genes.

关 键 词：家蚕核型多角体病毒 lef5基因 病毒复制 基因转录 

分 类 号：Q939.4[生物学—微生物学] Q78