家蚕核型多角体病毒orf61基因缺失对病毒复制和各时期基因转录的影响  

Effects of Bombyx mori Nucleopolyhedrovirus orf61 Gene Deletion on Viral Replication and Gene Transcription at Different Stages

作　　者：薛圣杰 李俊[1,2] 余海鹏 张媛媛 胡冬冰[1,2] 全滟平 于威 

机构地区：[1]浙江理工大学生命科学学院,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》2017年第6期943-951,共9页ACTA SERICOLOGICA SINICA

基　　金：浙江省自然科学基金项目(No.LY17C170006);国家高技术研究发展计划"863"项目(No.2011AA100603);浙江理工大学521人才培养计划项目

摘　　要：orf61是杆状病毒晚期表达的核心基因之一。为了研究家蚕核型多角体病毒(Bm NPV)orf61基因的功能,利用Red重组技术与Bac-to-Bac系统分别敲除和异位补回orf61基因,构建了orf61缺失型病毒orf61-ko-Bacmid以及补回型病毒orf61-reBacmid,然后分别转染家蚕培养细胞Bm N,调查orf61基因缺失对Bm NPV复制和不同时期基因转录的影响。检测orf61基因缺失型病毒转染Bm N细胞液上清中的病毒滴度为0,说明orf61基因缺失导致病毒不能形成有感染活性的芽生型病毒粒子(BV);荧光定量PCR分析orf61基因缺失会显著地降低病毒基因组的复制水平,进而导致病毒各个时期基因转录水平极显著下降(P<0.01)。此外,通过体外EMSA试验和构建双荧光素酶报告基因系统,检测ORF61蛋白对多角体蛋白基因polh启动子的调控作用,结果显示该蛋白质是polh基因转录的负调节因子。研究结果有助于深入阐释Bm NPV orf61基因在病毒基因组复制中的作用以及对极晚期表达的多角体蛋白基因polh的转录调控作用。Orf61 is one of the core late expression genes in baculovirus. In order to explore functions of Bombyx mori nucleopolyhedrovirus（ Bm NPV） orf61 gene,Red recombination technique and Bac-to-Bac system were used to knockout and rescue orf61 gene respectively for construction of the knockout bacmid（ orf61-ko-Bacmid） and the rescued bacmid（ orf61-re-Bacmid）. Consequently,these two bacmids were transfected into Bm N cells to explore the influence of orf61 gene deletion on viral replication and gene transcription at various stages. Our detection results showed that the titer of orf61 knockout virus in supernatant of transfected Bm N cells was 0,meaning that orf61 gene deletion had led to failure of the virus in forming infectious budded virus（ BV）. Fluorescent quantitative PCR assay showed that orf61 gene deletion significantly reduced the replication level of viral genome,which further led to an extremely significant reduction of viral gene transcription level at each stage（ P 0. 01）. Moreover,we utilized EMSA and dual-luciferase reporter assay system to detect the regulatory function of ORF61 protein on promoter of Bm NPV polh gene. The result showed that ORF61 is a repressor protein to polh gene. These results will facilitate further elucidation on functions of Bm NPV orf61 gene in viral replication and gene transcription and in transcriptional regulation of the very-late-expressed polh gene.

关 键 词：家蚕核型多角体病毒 orf61基因 病毒复制 基因转录调控 多角体蛋白基因启动子 

分 类 号：S884.51[农业科学—特种经济动物饲养]