【作者】 王晓锋；

【导师】 张传溪；

【作者基本信息】 浙江大学 ， 生化与分子生物学， 2012， 博士

【摘要】 杆状病毒(Baculovirus)是一类能够感染并最终杀死鳞翅目、膜翅目等农业害虫和双翅目蚊子的一类双链环状DNA病毒,其基因组大小为80-180kb,基因总量约为895个；该类病毒可用作生物农药、基因表达载体、基因治疗载体等,广泛应用于农业生产或医学研究等领域。目前已经有58种杆状病毒完成了基因组测序与注释,包括45种核型多角体病毒(NPV),13种颗粒体病毒(GV)；然而,仅有5种杆状病毒蛋白组成得到鉴定,而且全部为NPV病毒,鉴定蛋白多为ODV(?)蛋白,仅1种为BV (budded virus)蛋白。鉴于此,本研究选择菜粉蝶颗粒体病毒(PrGV,属GV病毒)为研究对象,重点分析该病毒基因组、ODV蛋白组成等,本论文主要结论如下：1. PrGV基因组分析该病毒基因组为闭合环状双链DNA,大小108,592bp,拥有120个ORFs,编码区占整个基因组91%；在120个预测的ORFs中,65个ORFs与granulin基因具有相同的转录方向,另55个ORFs的转录方向与其相反；PrGV基因组中最长ORF含有3402bp (ORF75, helicase-1),推测编码1133个氨基酸残基的蛋白；含有2个重复基因(ODV-E66a/b)；PrGV基因组存在基因间隔区和基因重叠现象,8个同源区分散在基因组不同位置。PrGV基因组存在杆状病毒科31种核心(保守)基因和鳞翅目杆状病毒31种辅助基因。PrGV基因组共线性分析揭示了该病毒与β-杆状病毒属基因组具有异常高的共线性,其中PrGV与云杉卷叶蛾颗粒体病毒(ChocGV)、杨扇舟蛾颗粒体病毒(ClanGV)、茶小卷叶蛾颗粒体病毒(AdorGV)等基因组共线性程度非常高,与八字地老虎颗粒体病毒(XecnGV)等基因组共线性程度相对较低；而与鳞翅目核型多角体病毒AcNPV共线性程度更低。蛋白质同源比对分析揭示了PrGV ORFs可分为四类：1)8个GV-特异基因这些基因同系物仅仅存在于GV基因组中,它们是orf4, orf12, orf28, orf34, orf55, orf95(LEF-3), orf103, orf112,表明这些保守基因是GV病毒结构与功能所必须；2)33个基因存在于GV基因组和其它病毒基因组,但在NPV基因组中并不存在,他们是orf2,orf5,orf18,orfl9,orf20(PEP1),orf24,orf25,orf27,orf31,orf35,orf36,orf37(Metalloproteinase),orf41,orf42.orf48(39K),orf54,orf62(FGF-1),orf63, orf67,orf83,orf86,orf89,ort94,orf97,orf102,ort104,orf105,orf106,ort109, orf111,orf114,orf115;3)4个基因仅存在于杆状病毒PrGV基因组中或其它物种中,它们是off09,orf32,orf53,oef117；4)75个基因为GVs和NPVs共享基因。基因组进化分析显示：PrGV基因组无论在LCB(Locally Collinear Blocks,局部共线区)数目、方向、排列顺序、长度等方面与ChocGV基因组高度相似,暗示了PrGV与ChocGV在基因组进化上可能经历着相似途径；PrGV与PsunGV, XeniGV,HearGV对应的LCBs相比,在LCB序列长度方面存在较大差异,推测3种病毒(PsunGV,XeniGV,HearGV)在长期的选择压力下,通过在LCB区域“扩充”基因以“增添”新功能来更好地拓展生存空间；PrGV与AcNPV基因组对应LCBs相比差异显著,揭示了两种病毒在基因组进化方面分歧较早；PrGV与CunniNPV基因组相比,两者的LCBs一致性最低,暗示了两者分歧时间更早,但都保留了“祖先”基因组某些基因。PrGV种系发生研究表明：PrGV与ChocGV在种系进化树上距离最近；13种β-杆状病毒聚类在一起,形成1个分支；在分支内6种病毒(HearGV,XeniGV, PsunGV,SpliGV AgseGV,PlxyGV)形成亚分支,7种病毒(AdorGV,PhopGV, CrleGV,CypoGV,ClanGV,ChocGV和PrGV)形成另1个亚分支。2.PrGV-ODV蛋白组分析利用3种质谱技术对rGV-ODV蛋白分析共鉴定47种蛋白,其中MALDI-TOF/TOF MS(matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight mass spectrometry:基质辅助激光解离串联飞行时间质谱)方法鉴定16种蛋白,LC-LTQ Velos(liquid chromatography-linear ion trap quadrupole Velos MS液相色谱-双压线性离子阱质谱)技术鉴定37种蛋白；而LTQ-Orbitrap (linear ion trap quadrupole-Oritrap MS:二维线性离子阱-轨道阱联用质谱)方法鉴定31种蛋白；47种蛋白中有11种被所有3种质谱技术所鉴定出；15种蛋白至少被2种质谱方法鉴定出；21种蛋白仅被1种质谱方法鉴定。在鉴定的47种ODV蛋白中,14种蛋白(P10,Pr21,Pr29,Pr35,Pr42,Pr54,P45/48,Pr83,Pr84,Pr89,Pr92,Prlll,Pr114和FGF3)为新鉴定的ODV蛋白,其中7种蛋白为β-杆状病毒特有蛋白(Pr35,Pr42,Pr54,Pr83,Pr84,Prlll和Pr114)。通过对鉴定的47种蛋白随机选择11种ORFs进行基因克隆、表达与多抗制备等试验,并对PrGV病毒蛋白进行免疫杂交分析,结果显示所有抗体均显示阳性信号,这进一步证实了11种蛋白在PrGV病毒中的存在,同时验证了质谱技术鉴定蛋白的可行性、可靠性。3.Pr42/Pr114基因分析基因组和蛋白组分析表明,Pr42和Pr114基因为β-杆状病毒特有基因(GV),两基因表达产物均为PrGV高丰度蛋白。生物信息学分析显示：Pr42基因编码803个氨基酸残基,预测分子量为93.5 kDa；该蛋白存在较为丰富的磷酸化位点(52个)和3个O-Glycosylated糖基化位点；Pr42为亲水性蛋白,不存在信号肽序列；进化树分析表明：Pr42与ClanGV gpO22蛋白同源性最高；Pr114基因编码323个氨基酸残基,预测分子量为36.9 kDa,该蛋白存在较为丰富的磷酸化位点(21个)、不存在O-Glycosylated糖基化位点；Pr114为亲水性蛋白,不存在信号肽序列；进化树分析表明该蛋白与ChocGV-ORF110蛋白同源性最高。RT-PCR分析表明,两种蛋白基因Pr42/Prll4的转录在经口感染48hpi就可以检测到,随后一直持续到96hpi；定量PCR分析表明：Pr42和Pr114基因在病毒感染6-96h均有转录,而在感染60h时相对转录水平均到达最高。综合生物信息学分析以及RT-PCR、定量PCR实验结果,推测Pr42/Pr114基因均为晚期表达基因,两蛋白功能可能涉及病毒结构组装,为PrGV重要的结构蛋白。更多还原

【Abstract】 The family Baculoviridae is a diverse group of rod-shaped, enveloped viruses with circular double-stranded DNA genomes ranging in size from 80 to 180 kb and its gene content is about 859. Baculoviruses could infect and finally kill insect pests of Lepidoptera, Hymenoptera and Mosquito of Diptera. Baculoviruses have been extensively used for insecticides and some are widely used as vectors in gene expression systems, some for gene therapy in medical study. Presently,58 baculovirus genomes have been sequenced, including 45 NPVs and 13 GVs; however, only 5 baculovirus proteins composition were identified from only NPVs, including 4 proteomes from ODV and only one proteome from BV. This study explored Pieris rapae Granulovirus (PrGV) genome structure and organization, ODV protein composition etc., and the results were as follows1. PrGV genome analysisPrGV genome size was 108,592 with 120 ORFs and coding region accounted for 91% of the whole genome. Of 120 putative ORFs,65 ORFs featured the same transcription direction with granulin and other 55 ORFs did the opposite direction; The longest ORF in PrGV genome had 3402bp (ORF75, helicase-1) and coding protein had 1133 amino acid residues; The genome has two repeative genes (ODV-E66a/b); PrGV genome featured non-transcription region, overlapping genes and 8 homologous regions were dispersed different positions in genome.31 core genes in all sequenced baculoviruses genomes and 31 conservative genes in Lepidoptera baculovirus genomes were detected in PrGV genome.Gene colinearity analysis disclosed that the PrGV ORFs featured high colinearity with the parallel parts ofβ-baculovirus. The highest colinearity occurred between PrGV and ChocGV, ClanGV, AdorGV genomes and the lower colinearity with XecnGV while the lowest with AcNPV.Blast P analysis represented the ORFs in PrGV were classified as follows:1) 8-GV unique genes, which only existed in GV-genomes. They were orf4, orf12, orf28, orf34, orf55, orf95(LEF-3), orf103, orf112.2) 33 ORFs existed in GV genome and other species but not in NPV genome, there were orf2, orf5, orf18, orf19, orf20(PEP1), orf24, orf25, orf27, orf31, orf35, orf36, orf37 (Metalloproteinase), orf41, orf42, orf48(39K), orf54, orf62(FGF-1), orf63, orf67, orf83, orf84, orf86, orf89, orf94, orf97, orf102, orf104, orf105, orf106, orf109, orf111, orf114, orf115; 3) 4 ORFs only existed in PrGV genome and other species but not in NPV genome, they were orf09, orf32, orf53, orf117.4) 75 ORFs shared with homolog with NPV genome.Genome evolution analysis showed that PrGV genome featured extremely high identity with ChocGV genome in whatever LCB (Locally Collinear Blocks) number, direction, arrangement order or size, disclosing that both viruses shared similar evolution traces. The lower identity occurred between PrGV genome and PsunGV, XeniGV, HearGV genomes for comparably big differences in LCB size, which implied that 3 viruses (PsunGV, XeniGV, HearGV) could expand genes to add new functions in order to expand their host limit during the long course of selection pressure. The big difference in LCBs occurred between PrGV and AcNPV, representing that both viruses featured earlier diverge in their individual evolution. The lowest identity in LCBs showed more earlier diverge occurred between PrGV and CuniNPV genome, however, both viruses inherited some ancestral genes.Phylogeny analysis disclosed that the closest genetical relationship occurred between PrGV and ChocGV. As expectedly,13β-baculoviruses cluster into one clade including 6 viruses (HearGV, XeniGV, PsunGV, SpliGV, AgseGV, PlxyGV) in one subclade and other 7 viruses (AdorGV, PhopGV, CrleGV, CypoGV, ClanGV, ChocGV, PrGV) in another subclade.2. PrGV-ODV proteome analysisForty seven proteins were identified by 3 MS approaches from PrGV-ODV. Of 47 proteins,16 proteins were identified by MALDI-TOF/TOF MS,37 proteins were done by LC-LTQ Velos MS while 31 proteins by LTQ-Orbitrap MS. Notably,11 proteins were identified by all 3 MS approaches,15 proteins were done by at least 2 MS approaches while 21 proteins by only one MS method.Of 47 proteins,14 proteins (P10, Pr21, Pr29, Pr35, Pr42, Pr54, P45/48, Pr83, Pr84, Pr89, Pr92, Pr111, Pr114 and FGF3) were regarded as newly-identified ones, including 7 GV-unique proteins (Pr35, Pr42, Pr54, Pr83, Pr84, Pr111 and Pr114). Immunoblotting analysis, using antisera prepared from 11 randomly selected recombinant, further confirmed that 11 proteins existed in PrGV-ODV, which disclosed the feasibility of MS approach for protein identification.3. Genes analysis of Pr42 and Pr114Bio-information analysis showed that Pr42 gene codes 803aa, putative MW(molecular weigh) is 93.5kDa; the gene product features 52 phosphorylation sites and 3 O-Glycosylated sites; Pr42 is hydrophilic protein with no signal peptide and phylogeny showed that Pr42 shared highest identity with ClanGV gp022; Pr114 gene codes 323aa, putative MW is 36.9kDa; the gene product features 21 phosphorylation sites and no O-Glycosylated sites; Pr114 is also hydrophilic protein with no signal peptide and phylogeny showed that Pr114 shared highest identity with ChocGV-ORF110.RT-PCR analysis represented that both Pr42 and Pr114 genes began to transcribe at 48h per os infection and did till to 96h. Realtime PCR analysis showed Pr42 and Prll4 transcripts could be detected at 6-96h per os infection, respectively. The relative transcripts level featured big differences during different infection phases.Combined bioinformatics, RT-PCR with real-time PCR analysis, Pr42/Prll4 maybe late expression genes and their genes products could function as structure proteins.更多还原