【作者】 吴萍；

【导师】 郭锡杰；

【作者基本信息】 中国农业科学院 ， 特种经济动物饲养， 2010， 博士

【摘要】 家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus, BmCPV)是养蚕业的主要病原之一,给养蚕业带来了巨大的经济损失。开展家蚕感染质型多角体病毒相关基因的研究,正确理解宿主与质型多角体病毒的应答与互作关系对质型多角体病的诊断及研发新的防治措施至关重要。本研究采用抑制消减杂交技术构建了家蚕感染BmCPV中肠和正常中肠正反向消减cDNA文库,测序分析共得到36个已知基因和20个新的ESTs序列。荧光定量PCR分析了3个上调基因(核糖体蛋白L11、铁蛋白和碱性核酸酶基因)及3个下调基因(抑制凋亡蛋白、丝氨酸蛋白酶及类胰蛋白酶基因)在感染BmCPV 6h、12h、24h、48h及72h后的相对转录水平。采用基因芯片技术比较分析了感染家蚕BmCPV的试验组与对照组中的差异表达基因。在感染24h、48h及72h后,分别得到了9个、51个及258个差异表达基因。KEGG通路分析表明,在感染48及72h后,大多数涉及到代谢通路的基因的表达水平发生了下调,所有涉及到核糖体通路和蛋白酶体通路的基因的表达水平均发生了上调。一些免疫相关基因如丝氨酸蛋白酶抑制剂、脂酶、热激蛋白、细胞色素P450、核糖体P0蛋白基因的表达水平发生了上调,具有氧化还原酶活性功能的基因其表达水平均发生了下调。研究表明家蚕感染BmCPV后,体内的氨基酸代谢水平受到破坏,宿主细胞内蛋白质合成降低,一些与转录翻译相关的基因为了满足病毒增殖的需求被高水平表达。另一方面,宿主细胞为抵制病毒的入侵可能启动了凋亡机制来清除被感染的细胞,病毒的入侵也诱导了一些与自身免疫相关的基因的表达上调。采用快速末端扩增技术首次克隆了编码家蚕泛素活化酶E1功能蛋白1基因(ubiquitin-activating enzyme E1- domain containing 1, UbE1DC1)及另一个未知功能的家蚕假定蛋白基因的全长cDNA,并对其氨基酸序列进行了生物信息学分析。UbE1DC1基因全长cDNA为1919 bp,包含100 bp的5’非翻译区和637 bp的3’非翻译区。开放阅读框1182 bp,共编码393个氨基酸。该蛋白含有一个THiF_MoeB_hesA_family结构域,这是一个ATP结合位点,属泛素活化酶E1家族。家蚕假定蛋白基因全长cDNA为486 bp,包含108 bp的5’非翻译区和153 bp的3’非翻译区。开放阅读框225 bp,共编码74个氨基酸,该蛋白含二次跨膜结构,整个多肽链表现为疏水性,可认为是疏水性蛋白。UbE1DC1及家蚕假定蛋白基因在丝腺、血液、脂肪体、生殖体及中肠中均可表达,荧光定量PCR结果表明,UbE1DC1在正常中肠中的表达水平显著高于感染中肠中的表达水平,是其9.78倍。推测当BmCPV入侵家蚕后,UbE1DC1的转录水平下调,使得感染细胞免受凋亡而进一步增殖。家蚕假定蛋白基因在感染BmCPV中肠中的表达水平显著高于正常中肠中的表达水平,是其6.28倍。上调机制尚不清楚。研究结果为从分子水平上阐明BmCPV对家蚕的致病机理提供了新的理论依据和研究方向,为进一步研究UbE1DC1及家蚕假定蛋白基因与BmCPV感染的关系奠定了基础。更多还原

【Abstract】 Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), one of the major viral pathogens for the silkworm, causes enormous damages to the sericultural industry. Studies on genes related to silkworm infected with BmCPV and understanding of the host response to Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is crucial to the diagnosis of BmCPV-caused silkworm disease and the development of new control measures.Suppression subtractive hybridization method was employed to construct forward and reverse subtractive libraries from the midguts of BmCPV-infected and normal silkworm larvae. Total of 36 genes and 20 novel ESTs were identified from two reciprocal subtractive libraries. The relative transcript level of three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and three down-regulated genes (serine protease, trypsin-like protease and inhibitor of apoptosis protein) at 6, 12, 24, 48 and 72h post-inoculation were analyzed by quantitative real-time PCR.Microarray analysis was employed to compare differentially expressed genes in the midguts of BmCPV-infected and normal silkworm larvae. At 24h, 48h and 72h post-inoculation, 9, 51 and 258 differentially expressed genes were identified respectively. KEGG pathways analysis indicated that at 48h and 72h post-inoculation, most genes related to metabolism pathways were down-regulated while expressions of genes involved in ribosome and proteasome pathway were all up-regulated. The expressions of several immune-related genes including serpin5, lipase, heat shock proteins, cytochrome P450s and ribosomal P0 protein were up-regulated. The expressions of genes grouped into oxidoreductases activity were all down-regulated.After the invasion of BmCPV, the function of digestion and absorption of midguts was damaged. This resulted in the decrease of the protein and amino acid metabolism in the silkworm. A large number of genes used for translation were over-expressed to meet the needs of virus replication. On the other hand, the host cells may startup apoptosis program to defense the proliferation of BmCPV. Invasion of BmCPV also induced the expressions of several immune-related genes.We firstly cloned the full length cDNA which encodes the ubiquitin-activating enzyme E1-domain containing 1 (UbE1DC1) and a hypothetic protein gene of the silkworm by using rapid amplification of cDNA ends (RACE). The full-length cDNA of UbE1DC1 gene is 1919 bp, consisting of a 100bp 5’untranslated region, a 637 bp 3’untranslated region and a 1182 bp open reading frame (ORF), encoding 393 amino acids. The protein contained the THiF_MoeB_hesA_family domain, an ATP binding site, which is belonged to the family of ubiquitin-activating enzyme E1. The full-length cDNA of hypothetic protein gene is 486 bp, consisting of a 108bp 5’untranslated region, a 153bp 3’untranslated region and a 225bp open reading frame (ORF), encoding 74 amino acids. RT-PCR analysis of the silkworm tissues silk gland, hemocyte, fat body, gonad and midgut revealed that the UbE1DC1 and hypothetic gene were expressed in all the five tissues. The quantitative real-time polymerase chain reaction analysis indicated that the relative expression of UbE1DC1 in the normal midgut was approximately as 9.78 fold of that in the BmCPV-infected midgut. It is implicated that the down-regulation of UbE1DC1 could prevent the infected cells from apoptosis. The relative transcript of hypothetic protein gene in the infected midgut was 6.28 fold than that in the normal midgut. The mechanism of its up-regulation is not clear.Our results provided not only new clues for investigating the molecular mechanism of BmCPV infection but also the theoretical basis for the furthe’[r study on the function of UbE1DC1 and hypothetic protein gene of Bombyx mori.更多还原