【作者】 唐平；

【导师】 张志芳；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 杆状病毒具有闭合环状双链DNA基因组，是一类主要感染鳞翅目、膜翅目和双翅目昆虫的病原物，杆状病毒作为生物杀虫剂具有对人畜安全，不造成环境污染，宿主专一性强，能有效控制田间害虫种群密度等优点。本文对一株分离自棉铃虫的核型多角体病毒进行电镜观察，表明其为多粒包埋型的核型多角体病毒，昆虫感染实验表明其与棉铃虫单粒包埋型核型多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus，HearSNPV)有不同的宿主域，细胞感染实验表明其与HearSNPV有不同的病理效应，因此对其基因组进行了分析，并与其它杆状病毒基因组进行了比较。对一株分离自棉铃虫的病毒纯化后进行电镜观察，发现其呈不规则多角体型，包涵体中有多个包含病毒核衣壳的囊膜，每个病毒囊膜内含有2个以上核衣壳，符合多粒包埋型核型多角体病毒特征，将其命名为棉铃虫多粒包埋型核型多角体病毒（Helicoverpa armigera multinucleocapsidnucleopolyhedrovirus，HearMNPV）。荧光定量PCR结果分析表明HearMNPV的DNA在棉铃虫体内复制经历了减少期、隐晦期、增长期及稳定期４个时相，表明其宿主体内复制良好。HearMNPV对其它昆虫的感染性实验表明它对东方粘虫有较好的感染性，但不能感染斜纹夜蛾及甜菜夜蛾，而HearSNPV不能感染东方粘虫，表明两者有着不同的宿主域。HearMNPV对Hz-AM1细胞系感染后没有多角体产生，也不显示明显的细胞病理效应；对棉铃虫胚胎细胞系QB-Ha-E-5细胞系感染后有大量多角体产生，呈现较好的感染性。Hz-AM1是HearSNPV的敏感细胞系,用源自HearSNPV的HaBacmid-egfp对QB-Ha-E-5细胞系感染，细胞感染后有明显的荧光产生，表明HearMNPV和HearSNPV对宿主细胞感染性存在不同。为进一步分析HearMNPV，将Sau3AⅠ部分酶切HearMNPV基因组DNA，SalⅠ完全酶切pUC19载体，以Klenow酶分别对回收的片断和载体进行半补齐反应，经连接、转化后,得到用半补齐法构建的覆盖率大于99.9％的HearMNPV基因组文库。对HearMNPV基因组全序列进行测定并分析表明基因组的大小为154,196bp，G+C含量为40.07%，推测含有162个与相邻的ORF有最小重叠且大于150bp的ORF，占基因组全序列的90.16%，4个同源重复区等非编码区序列占整个基因组的9.84%。基因对等图表明HearMNPV与HearSNPV共线性较低，HearMNPV基因组结构与披肩粘虫核型多角体病毒B株（Mamestraconfigurata nucleopolyhedrovirus-B，MacoNPV-B)有很好的共线性；系统进化分析表明HearMNPV与HearSNPV不在同一簇中，而与MacoNPV-B存在同一簇中。HearMNPV与HearSNPV在基因组的大小、内容，同源基因的排列、一致性上存在着显著的差别，结合昆虫宿主域实验确定HearMNPV是一株不同于棉铃虫单粒包埋型核型多角体病毒(HearSNPV)的新病毒种。与MacoNPV-B基因组结构相比，HearMNPV缺失了包含ORF54、55、56、57、58的片断，长度约为5.4kb，其基因组中HearMNPV orf66、orf17、bro基因、hr序列与MacoNPV-B存在明显的不同，表明其与MacoNPV-B的亲缘关系然很近，但在基因组的结构上存在明显的差异。更多还原

【Abstract】 The baculoviruses is a family of invertebrate viruses with large, circular, and double-stranded DNAgenomes. They are pathogenic to arthropods, mainly insects of the orders Lepidoptera, Hymenoptera,and Diptera. The baculoviruses is the most commonly investigated with regard to its development as amicrobial insecticide due to its favorable characteristics such as safety to the environment, humans, othervertebrates, plants, and natural enemies of pests. Consequently, the baculoviruses are ideal controlagents to be used in integrated pest management (IPM) programs in agriculture, forests.In this study, anew nucleopolyhedrovirus isolated from H.armigera was observed by electron microscope (EM),suggesting it was multinucleocapsid NPV.Experimental infection of insect larvae indicated that hostrange of HearMNPV was different from that of HearSNPV and that the cytopathological effect ofHearMNPV differed from that of HearSNPV. This report describes the sequence and organization of theHearMNPV genome and compares it with sequence data from other baculoviruses, such as HearSNPVand MacoNPV-B.The purified occlusion bodies (OBs) of NPV origin from the infected cotton bollworm have irregularshape by scanning electron microscope. Multiple rod-shaped virions were embedded in each OB withmultiple nucleocapsid packaged within the envelope of the virion by transmission electron microscope.These results indicated that it belong to typical multiple NPV.Therefore the isolate is denominated asHelicoverpa armigera multinucleocapsid nucleopolyhedrovirus(HearMNPV).The quantity of HearMNPV viral DNA, relative to host actin gene copy number, in infected larvae,included decreasing phase, latent phase, exponential phase and stationary phase, the results suggestedthat the cotton bollworm was infected by HearMNPV well. Experimental infection of insect larvaeshowed that HearMNPV can infect the eastern armyworm (Pseudaletia separate) but cannot infect eitherthe cotton leaf worm (Spodoptera litura) or the beet army worm (Spodoptera exigua). In contrast,HearSNPV cannot infect Pseudaletia separate. These results indicated that the host range of HearMNPVdiffered from that of HearSNPV. Moreover, HearMNPV-infected Hz-AM1cells produced no polyhedraand showed no typical cytopathic effects (CPE). However, HearMNPV-infected QB-Ha-E-5cellsproduced polyhedra. It was previously reported that Hz-AM1cells were permissive to HearSNPV, theQB-Ha-E-5cells were infected by HaBacmid-egfp which were constructed from HearSNPV, greenfluorescence were observed by fluorescence microscopy. These results indicated that the host range andcells infected with HearMNPV differed from those infected with HearSNPV.To better understand HearMNPV, the genomic DNA of HearMNPV was partially digested bySau3AⅠ, the plasmid vector pUC19was fully digested by SalⅠ, and subsequently filled in by theKlenow fragment respectively. After ligation and transformation, a genomic library of HearMNPV withthe representation of more than99.9%was constructed by “partial filling-in” method.The HearMNPV genome consists of154,196base pairs, with a G+C content of40.07%. A total of162putative ORFs were detected in the HearMNPV genome which represented90.16%of the genome.The remaining9.84%constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of MacoNPV-B,but distinguished from HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-Bsuggested that HearMNPV has a deletion of a5.4-kb fragment which contained5ORFs. In addition,HearMNPV orf66、 bros、 hrs were different with the corresponding parts in MacoNPV-Bgenome.HearMNPV can replicate in vivo and in vitro of Helicoverpa armigera, which is a new isolateNPV distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but with adistinct genomic structure, content, and organization.更多还原