【作者】 田彩红；

【导师】 张传溪；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 到目前为止，有48种昆虫杆状病毒的基因组被完全测序，而家蚕核型多角体病毒(Bombyx mori nucloepolyhedrivirus，BmNPV)作为杆状病毒的模式病毒之一，得到了越来越广泛的研究和应用。目前，有许多基因的功能已了解清楚，例如Bm67、Bm56、Bm9等，但仍有许多基因的功能尚是未知。本研究中，选取了orf41和orf51两个功能未知基因为研究对象，从基因的转录、表达、亚细胞定位、病毒结构定位、基因缺失等方面研究基因的基本特性及其初步功能，从而丰富杆状病毒分子生物学理论，并为下一步深入分析基因功能和相关病毒基因工程研究提供基础。本研究的主要结论如下：1．BmNPV orf41(Bm41)基因分析Bm41位于BmNPV(T3株)基因组39204-39788nt，读码框全长585bp，编码194个氨基酸，预测分子量约为23.3kDa。在Bm41起始密码子ATG上游146nt处有一杆状病毒晚期转录基序CAGT。用BLASTP工具搜索GenBank和SWISS-PROT数据库发现，在25种目前已经测序的鳞翅目基因组中存在Bm41的同源序列，包括GroupⅠNPV和GroupⅡNPV，但在于鳞翅目GVs和其它杆状病毒并未发现。因而推测，Bm41及它的同源基因是鳞翅目昆虫核型多角体病毒特有基因，在其它杆状病毒寄主系统中，这个基因可能是非必需的或细胞内因子可以补偿这种缺陷。为了进一步研究Bm41在病毒侵染循环中的作用，利用ET重组系统成功构建了Bm41的突变病毒，进而利用Bac-to-Bac系统构建了一个在多角体位点重新补回Bm41的补回病毒。电镜分析表明，敲除Bm41影响了病毒核衣壳的形成以至进一步影响了成熟的ODV形成多角体。Western blot分析表明，Bm41既不是ODV也不是BV的结构蛋白的组成部分。细胞内定位表明，Bm41可能在细胞核和细胞质均有功能。转录和表达时相表明Bm41是一个早期转录的基因。用突变的病毒感染细胞样品进行western blot分析表明，一些晚期基因的表达受到了影响，ODV-EC27的表达受到了明显的下调，多角体基因的表达则受到了抑制，不再表达。生物测定分析表明，与突变前野生病毒相比，突变的Bm41病毒延长了宿主昆虫的LT50，也提高了宿主昆虫的LD50。BV滴度分析表明，Bm41的突变大大降低了BV在细胞内的产量。综上所述，BmNPV orf41是一个早期基因，编码的蛋白与结构蛋白无关。Bm41的突变影响了病毒DNA复制并在细胞和虫体内产生了很低产量的BV。而且，Bm41缺少的病毒对宿主昆虫的感染力降低并延长了对宿主昆虫的致死时间。电镜分析表明，Bm41的突变影响了核衣壳的形成。因此推测，Bm41尽管不是病毒复制的必需基因，但是在BV和ODV形成方面均起着重要作用，且是影响病毒在体内和体外感染力的重要基因。2．BmNPV orf51(Bm51)基因分析Bm51位于BmNPV(T3株)基因组45935-46402nt，读码框全长468bp，编码一个长155个氨基酸残基的蛋白，预测分子量为18.5kDa。在Bm51起始密码子ATG上游26nt处有1个杆状病毒晚期转录基序CAGT。RT-PCR分析表明，Bm51在4.5h p．i．开始转录。利用大肠杆菌表达系统表达了GST-Bm51融合蛋白并利用该蛋白制备了多克隆抗血清。利用此抗血清进行Bm51的表达时相分析发现，Bm51的表达产物在6h p．i．被检测出来。以上试验结果表明，Bm51是一个杆状病毒早期表达基因。利用抗Bm51的特异性抗体，对提取纯化的BV和ODV进行了westernblot分析，在提纯的BV泳道中出现了的23 kDa阳性条带。在ODV中没有检测出阳性条带。因此Bm51蛋白是BV特异性的结构蛋白。为了精确确定Bm51在BV中的定位。进一步将BV分离为BV核衣壳蛋白(BV-NC)和BV囊膜蛋白(BV-E)。利用Bm51抗体去杂交BV-NC和BV-E，发现只能在BV囊膜蛋白(BV-E)上杂交到阳性条带，而在BV-NC并没有任何条带。说明Bm51基因编码了与BV核衣壳相关的结构蛋白。为了对Bm51蛋白进行亚细胞定位，在BmNPV T3株病毒感染BmN细胞后72h，以制备抗Bm51的抗体为一抗，以protein G-EGFP为二抗进行细胞免疫定位。同时，加入细胞核特异结合染料DAPI以区分细胞核、质的位置。荧光显示，病毒位于细胞核和细胞质中。总之，Bm51是一个迟早期基因，编码一个BV囊膜结构蛋白。更多还原

【Abstract】 Until now, the genomes of 48 baculoviruses have been sequenced. Bombyx morinucleopolyhedrovirus（BmNPV） is one of the most studied baculoviruses; ManyBmNPV genes including Bm67、Bm56、Bm9, have been characterized; however,some genes remain unclear. In this study, two BmNPV genes, Bm41 and Bm51,conseved in Lepidoptera baculoviruses are characterized. The gene transcription, geneexpression, sub-cellular location, and gene disruption were adopted to characterizethese two genes. The object of this study is to enhance our understanding ofbaculovirus molecular biology. The main results are as following:1. Functional analysis of BmNPV orf41（Bm41）Bombyx mori nucleopolyhedrovirus（BmNPV） ORF41（Bm41）, homologous toAc52, is a gene present in most lepidopteran nucleopolyhedroviruses. The Bm41 islocated at 39204-39788nt in the genome of BmNPV T3 strain. Its ORF are 585bp inlength and is predicted to encode a 194 amino acid peptide with a deduced molecularweight of 23.3kDa.Bm41 transcripts and encoded protein in BmNPV-infected cells can be detectedfrom 3 and 6 h post-infection, respectively. Immunoassays have shown that Bm41 isnot a viral structural protein and is detected in both the nuclei and cytoplasm ofinfected cells. A Bm41-disrupted virus(vBm^De) and a repaired virus(vBm^Re) weregenerated to investigate the function of Bm41. The results showed that Bm41 wasessential for viral replication, and the disruption of Bm41 resulted in a much lowerviral titer. Transmission electron microscopy revealed that disruption of Bm41affected normal nucleocapsid envelopment and polyhedra formation in the nucleus.The disruption of Bm41 might severely affect odv-ec27 and polyhedrin expression.The disrupted virus reduced BmNPV infectivity in an LD50 bioassay and took 18-23h longer to kill larvae than wild-type virus in an LT50 bioassay.2. Functional analysis of BmNPV orf51（Bm51）BmNPV ORF 51（Bm51） is a gene present in many lepidopteran NPVs. TheBm51 is located at 45,935-46,402nt in the genome of BmNPV T3 strain. Its ORF are468 bp in length and is predicted to encode a 155 amino acid peptide with a deducedmolecular weight of 18.5kDa. Transcripts of Bm51 were detected from 4.5 through 72hour post infection（h p.i.） by RT-PCR. The corresponding protein was detected from6 to 72 h p.i. in BmNPV-infected BmN cells by western blot analysis using apolyclonal antibody against Bm51. Western blot assay of occlusion-derived virus andbudded virus（BV） preparations revealed that Bm51 encodes a 23-kDa structuralprotein that is associated with BV and is located in the envelope fraction of buddedvirions. The protein was temporarily called BV-E23. In addition immunofluorescencemicroscopy demonstrated that the protein was present within the cytoplasm and nucleiin virus-infected cells. In conclusion, the available data suggest that Bm51 is afunctional ORF of BmNPV and encodes a protein expressed in the early stage of theinfection cycle that is associated with the BV envelope.更多还原