中国棉铃虫核多角体病毒（HearNPV）orf107，orf135及pkip基因功能的研究
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摘要
棉铃虫核多角体病毒(HearNPV)是在我国农业生物防治中广泛应用的重要病毒，我们在前期测定棉铃虫病毒基因组序列的基础上，全面开展了HearNPV基因的功能研究。本论文对HearNPV中的三个基因：Ha107，Ha135和Ha130(pkip)基因进行了转录、表达分析、定位等研究，并通过基因缺失初步研究了它们的功能。
第二章的研究发现Ha107预测编码一个51kDa的蛋白。转录分析表明，Ha107在HearNPV感染的HzAM1昆虫细胞中具有两个转录起始位点，一个位于Ha106(superoxide dismutase，sod)的上游，另一个位于Ha107上游。Western blot结果显示，HA107蛋白在HearNPV感染的细胞中具有两种形式，主要形式为48kDa，另一个少量形式为51kDa。Western blot结果同时显示HA107是BV和ODV的核衣壳结构组分。缺失Ha107增加了出芽病毒粒子(BV)的感染性。电镜观察显示Ha107的缺失对包涵体的形成并无显著影响。这些结果表明Ha107编码一个非必需的结构蛋白；对该基因的缺失会导致BV感染性增强。
Ha135预测编码一个23kDa的蛋白。第三章描述了对Ha135的缺失分析和功能鉴定。3'RACE结果表明：在病毒感染后12小时细胞RNA样品中最先检测到Ha135基因的mRNA，并持续到感染末期；并确定了转录的起始和终止位点。Western blot结果显示Ha135编码一个23kDa非结构蛋白。当我们将该蛋白C端与eGFP蛋白融合后，瞬时转染结果显示HA135蛋白定位在细胞核中，并聚集成块状。凝胶分子筛层析以及化学交联实验结果表明HA135蛋白在体外主要以二聚体形式存在。Ha135的缺失并没有显著影响BV生长曲线。Ha135缺失的病毒对棉铃虫幼虫仍具有感染性。
Ha130基因全长510bp，预测编码一个20kDa的蛋白激酶相互作用蛋白(PKIP)。第四章对HearNPV PKIP进行了研究。利用3’RACE对HearNPVpkip的转录进行分析表明：在病毒感染后8小时细胞RNA样品中最先检测到pkip基因的mRNA，并持续到感染末期。Western blot分析显示，pkip的表达产物大小为20kDa，与理论分子量接近。亚细胞定位实验显示，单独表达的PKIP-GFP融合蛋白主要分布在被转染的HzAM1细胞的细胞质中，在病毒超感染后，融合蛋白在核内的分布增多。缺失分析表明pkip不是HearNPV感染的必需基因。
Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is an important virusthat has been widely used in pest control in China. After genome sequence of theHearNPV, we are now focusing on elucidation of different genes of the virus. Inthis thesis, three of the ORFs, Hal07, Ha135 and Ha130 (pkip) were analyzed fortheir transcription, expression and localization. Their functions were studied bymaking knockout recombinant viruses.
Ha107 of HearNPV encodes a putative protein of 51 kDa with homologs in afew groupⅡNPVs and a GV. Functional analysis of this ORF is described inChapter 2. Ha107 was transcribed in infected HzAM1 insect cells aspolyadenylated transcripts initiated at two distinct locations. By Western blotanalysis two forms of the HA107 protein were detected in HearNPV infected cells,a major form of 48 kDa and a minor form of 51 kDa. The HA107 protein was foundin the nucleocapsid fraction of both budded virions (BVs) and occlusion-derivedvirions (ODVs). BV infectivity of HaBac△Ha107-eGFP-PH (Ha107 knockout virus)was much higher than that of 'control' virus and a Ha107-repair virus. Electronmicroscope analysis showed that the formation of occlusion bodies was notaffected by the deletion of Ha107. Our data indicate that Ha107 encodes anon-essential structural protein of HearNPV and that the deletion of this geneimproves the BV infectivity. transcribed as a polyadenylated transcript from 12 h post-infection in infected hostcells. Western blot analysis demonstrated the Ha135 encodes a 23 kDa protein,which was not present in budded virions (BV) or occlusion derived virions (ODV).When fused with green fluorescence protein, HA135 protein is predominantlydetected in the nuclei in host cells, with distinctive aggregates. Gel filtrationanalysis demonstrated that HA135 protein forms predominantly dimmer in vitro.Knockout Ha135 had no significant influence on polyhedra formation, BV growthcurve and ODV infectivity.
The protein kinase interacting protein (pkip) gene is a consensus gene amongnucleopolyhedrovirus. In Chapter 4, the function of HearNPV pkip wasinvestigated. 3'RACE analysis demonstrated that HearNPV pkip was transcriptedas polyadenylated transcripts in HzAM1 cells from 8 h post infection. By Westernblotting, the product of HearNPV PKIP was found as a 20 kDa protein in infectedcells, close to the theoretical molecular weight. Upon viral infection, PKIP-GFPfusion protein was observed primarily to be located in nucleus. Deletion ofHearNPV pkip gene showed no impair to budded virus production and occlussionbody formation. Thus, we conclude that the pkip gene is not essential forHearNPV infection.
第二章的研究发现Ha107预测编码一个51kDa的蛋白。转录分析表明，Ha107在HearNPV感染的HzAM1昆虫细胞中具有两个转录起始位点，一个位于Ha106(superoxide dismutase，sod)的上游，另一个位于Ha107上游。Western blot结果显示，HA107蛋白在HearNPV感染的细胞中具有两种形式，主要形式为48kDa，另一个少量形式为51kDa。Western blot结果同时显示HA107是BV和ODV的核衣壳结构组分。缺失Ha107增加了出芽病毒粒子(BV)的感染性。电镜观察显示Ha107的缺失对包涵体的形成并无显著影响。这些结果表明Ha107编码一个非必需的结构蛋白；对该基因的缺失会导致BV感染性增强。
Ha135预测编码一个23kDa的蛋白。第三章描述了对Ha135的缺失分析和功能鉴定。3'RACE结果表明：在病毒感染后12小时细胞RNA样品中最先检测到Ha135基因的mRNA，并持续到感染末期；并确定了转录的起始和终止位点。Western blot结果显示Ha135编码一个23kDa非结构蛋白。当我们将该蛋白C端与eGFP蛋白融合后，瞬时转染结果显示HA135蛋白定位在细胞核中，并聚集成块状。凝胶分子筛层析以及化学交联实验结果表明HA135蛋白在体外主要以二聚体形式存在。Ha135的缺失并没有显著影响BV生长曲线。Ha135缺失的病毒对棉铃虫幼虫仍具有感染性。
Ha130基因全长510bp，预测编码一个20kDa的蛋白激酶相互作用蛋白(PKIP)。第四章对HearNPV PKIP进行了研究。利用3’RACE对HearNPVpkip的转录进行分析表明：在病毒感染后8小时细胞RNA样品中最先检测到pkip基因的mRNA，并持续到感染末期。Western blot分析显示，pkip的表达产物大小为20kDa，与理论分子量接近。亚细胞定位实验显示，单独表达的PKIP-GFP融合蛋白主要分布在被转染的HzAM1细胞的细胞质中，在病毒超感染后，融合蛋白在核内的分布增多。缺失分析表明pkip不是HearNPV感染的必需基因。
Helicoverpa armigera nucleopolyhedrovirus (HearNPV) is an important virusthat has been widely used in pest control in China. After genome sequence of theHearNPV, we are now focusing on elucidation of different genes of the virus. Inthis thesis, three of the ORFs, Hal07, Ha135 and Ha130 (pkip) were analyzed fortheir transcription, expression and localization. Their functions were studied bymaking knockout recombinant viruses.
Ha107 of HearNPV encodes a putative protein of 51 kDa with homologs in afew groupⅡNPVs and a GV. Functional analysis of this ORF is described inChapter 2. Ha107 was transcribed in infected HzAM1 insect cells aspolyadenylated transcripts initiated at two distinct locations. By Western blotanalysis two forms of the HA107 protein were detected in HearNPV infected cells,a major form of 48 kDa and a minor form of 51 kDa. The HA107 protein was foundin the nucleocapsid fraction of both budded virions (BVs) and occlusion-derivedvirions (ODVs). BV infectivity of HaBac△Ha107-eGFP-PH (Ha107 knockout virus)was much higher than that of 'control' virus and a Ha107-repair virus. Electronmicroscope analysis showed that the formation of occlusion bodies was notaffected by the deletion of Ha107. Our data indicate that Ha107 encodes anon-essential structural protein of HearNPV and that the deletion of this geneimproves the BV infectivity. transcribed as a polyadenylated transcript from 12 h post-infection in infected hostcells. Western blot analysis demonstrated the Ha135 encodes a 23 kDa protein,which was not present in budded virions (BV) or occlusion derived virions (ODV).When fused with green fluorescence protein, HA135 protein is predominantlydetected in the nuclei in host cells, with distinctive aggregates. Gel filtrationanalysis demonstrated that HA135 protein forms predominantly dimmer in vitro.Knockout Ha135 had no significant influence on polyhedra formation, BV growthcurve and ODV infectivity.
The protein kinase interacting protein (pkip) gene is a consensus gene amongnucleopolyhedrovirus. In Chapter 4, the function of HearNPV pkip wasinvestigated. 3'RACE analysis demonstrated that HearNPV pkip was transcriptedas polyadenylated transcripts in HzAM1 cells from 8 h post infection. By Westernblotting, the product of HearNPV PKIP was found as a 20 kDa protein in infectedcells, close to the theoretical molecular weight. Upon viral infection, PKIP-GFPfusion protein was observed primarily to be located in nucleus. Deletion ofHearNPV pkip gene showed no impair to budded virus production and occlussionbody formation. Thus, we conclude that the pkip gene is not essential forHearNPV infection.
引文
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83．孟小林，徐进平，王健，鲁伟．我国病毒杀虫剂研究与应用新进展。植保技术与推广2002，22：38-39
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