【作者】 周阳；

【导师】 陈克平；

【作者基本信息】 江苏大学 ， 食品科学， 2010， 博士

【摘要】 家蚕是重要的经济昆虫,也是家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)的天然宿主。家蚕核型多角体病毒是引起家蚕病毒严重感染的关键病原之一,病毒与宿主的相互博弈一直是研究者们关注的问题。家蚕抗NPV机制至今尚未完全明确,但究其病因可归因于宿主与致病病毒两方面因素。本研究以BmNPV核心基因Bm5为研究对象,从基因的转录、表达、亚细胞定位、病毒结构定位和基因缺失等方面,研究基因的基本特性及其功能。同时使用家蚕高密度Oligo表达谱芯片所提供了相关基因的基因组学的证据,突破了以往单个基因孤立研究的局限,在全基因组范围内筛选家蚕抗NPV相关基因,对筛选出基因进行GO和Pathway分析,并进一步用半定量PCR和荧光定量PCR进行验证,探讨家蚕抗NPV的途径和机理。论文主要结论如下：1.BmNPV的orf5(Bm5)位于基因组4,605--5,600 nt,全长993 bp,编码一个含331个氨基酸残基的蛋白,预测分子量约为39.3 kDa,Bm5是AcMNPV orf13的同源序列。在Bm5基因ATG上游存在杆状病毒晚期转录基序ATAAG,在终止密码子TAA下游发现转录终止信号AATAAA,暗示了该基因是晚期表达基因。根据Bm5基因序列设计引物,分别以BmNP基因组DNA为模板进行PCR扩增,将得到的基因克隆至原核表达载体pET30a(+),在原核细胞中实现了基因的融合表达。纯化的蛋白免疫新西兰大白兔,制备了BM5蛋白的多克隆抗体。2. RT-PCR分析发现,Bm5在病毒感染宿主细胞12 h开始转录,直到72 h。利用抗体进行Western blot分析,发现Bm5的表达产物在病毒感染宿主细胞后24 h被检测出来,一直持续72 h都有表达,大小为39.3kDa,与预测的大小一致,说明Bm5是一个晚期表达基因,而且没有转录后修饰。Western blot方法检测分离出的BV、ODV粒子,表明BM5不是病毒粒子的结构蛋白。利用抗体检测发现BM5定位在细胞质中。3.利用Red重组系统在大肠杆菌中成功地对BmNPV的Bm5基因进行了快速定点敲除。并BmNPV/Bac-to-Bac系统和Red重组系统结合在一起,对敲除了Bm5基因的BmNPV:进行了拯救。分别构建了野生型病毒vBm5-Wt、Bm5基因缺失病毒vBm5-Ko和Bm5基因拯救病毒vBm5-Re。4.应用基因芯片技术在家蚕抗NPV品系BC8、感性品系306中筛查抗性相关基因,结果发现93个差异表达基因其中表达上调基因44个,下调基因49个。GO分析表明93个基因中51个其中24个上调基因、27个下调基因具有功能注释。GO分析结果显示分子功能方面主要有酶活性、氧化还原活性、酶绑定、结构成分、转运活性等；生物进程方面主要有新陈代谢、蛋白质水解、电子转移、蛋白质定位、转移、蛋白质生物等功能；细胞组分方面主要有细胞内、膜核糖体、胞外区等。Pathway分析表明有新陈代谢、果糖和甘露糖代谢、丙酸代谢、核糖体、氧化磷酸化、磷酸戊糖途径、糖酵解/糖异和柠檬酸循环(TCA循环)等通路。5.为了验证芯片筛选出差异表达基因,从93个差异基因挑选14个表达量差异大基因(9个上调基因、5个下调基因)进行RT-PCR,依据结果再从14个基因中挑选10个差异基因(7个上调基因、3个下调基因)进行荧光定量PCR。结果表明了7个上调基因中5个基因在NB、BC8各时间点的表达量高于306,鉴定为抗性相关上调基因,2个基因在BC8表达量高于NB、306。3个下调基因在306的表达量要高于NB、BC8,鉴定为抗性相关下调基因。表明荧光定量PCR试验结果与芯片结果具有良好的一致性,进一步证实了芯片结果的可靠性。更多还原

【Abstract】 Silkworm is an important economic insect. BmNPV (Bombyx mori L nucleopolyhedrovirus) is a key pathogeny to silkworm with highly infective. And the reciprocity between virus and host is an important issue which was paid many attentions by researchers all the time. Mechanism associated with BmNPV resistance has not yet entirely clear, but it could be attributed to both host and viral.In this study, a conserved gene (Bm5) in Lepidoptera baculoviruses was characterized from the transcription、expression、cellular location and knockout. The object of this study is to enhance our understanding of baculovirus molecular biology. We also screened genes associated with BmNPV resistance by using the Bombyx mori high-density Oligo microarray. The Microarray provided the whole genomic information of Bombyx mori, breaking the research limitations of single gene. In addition, we evaluated the datas by GO and Pathway, and further identified differentially expressed genes by using semi-quantitative PCR and fluorescence quantitative PCR. We hoped to elucidate the resistance mechanism. The results are as following:1. Orf5(Bm5) was located at nt 4,605-5,600 of BmNPV genome, containing 993 bp and coding 331 aa with a predicted molecular weight of 39.3 kDa. Bm5 was the homology sequence of AcMNPV orf13. The late transcription motif of baculoviridae, ATAAG was located at the upstream of ATG. And the transcription terminal signal, AATAAA was found at the downstream of the terminal codon TAA. Two primers were designed as the Bm5 gene sequence and used for PCR amplification. PCR products were cloned into pET30a(+) vector and Bm5 were expressed as fusion protein. The purified fusion proteins were injected into New Zealand white rabbits to harvest polyclonal antibodies. Western blot analysis of extracts from BmNPV-infected BmN cells revealed a specific polypeptide with an apparent size of 39.3 kDa when using the BM5 antiserum.2. The RT-PCR resulted showed that Bm5 gene was transcribed in the infected cells from 12 to 72 h. Bm5 fusion protein was expressed using the E. coli expression system and polyclonal antibodies were harvested. The western blot analysis showed that Bm5 was detected in the infected cells from 24 to 72 h, which further suggesting that Bm5 is a late gene. The detected BM5 had a molecular weight of 39.3 kDa, same as the predicted one, which suggesting that BM5 have no post-transcription modification. BM5 was not detected either in budded viruses or occlusion-derived virus. The sub-cellular localization of the BM5 proteins was investigated by immunofluorescence. The fluorescence was concentrated on the cytoplasm and no fluorescence was detected in the nucleoplasm.3. Bm5 was rapidly disrupted in E.cori by Red recombinant system. BmNPV which disrupted Bm5 was rescued by the BmNPV/Bac to Bac system and Red system. vBm5-Wt、vBm5-Ko and vBm5-Re viruses were constructed respectively. The results indicated the BmNPV Bac-to-Bac expression system can effectively express foreign gene.4. We screened 93 genes (44 up-regulation genes、49 down-regulation genes) associated with BmNPV resistance by using the Bombyx mori high-density Oligo microarray. The results of microarray were analyzed by Gene Ontology (2.0 version), which showed that 51 gene (24 up-regulation genes、27 down-regulation genes) have functional annotations. Molecular function of GO included enzyme activity、oxidoreductase activity、binding、structural constituen、transporter activity. Biological process included metabolism proteolysis、electron transport、protein targeting、transport、protein biosynthesis. Cellular components included intracellular、membrane、ribosome、extracellular region. Results of Pathway analysis included metabolism、fructose and mannose metabolism、acid metabolism、ribosome、oxidative phosphorylation、pentose phosphate pathway、glycolysis/gluconeogenesis、citric acid cycle (TCA cycle) and so on.5. We identified 10 differentially expressed genes (7 up-regulation、3 down-regulation) by Semi-quantitative PCR and fluorescence quantitative PCR. Results Showed the expression levels in 5 of 7 up-regulation genes were higher in NB and BC8 than in 306, which identified as up-regulation genes associated with BmNPV resistance,2 of 7 up-regulation genes were higher in BC8 than in NB. The expression levels in all 3 down-regulation genes were higher in 306 than in NB and BC8, which identified as down-regulation genes associated with BmNPV resistance. The results further confirmed the reliability of the chip更多还原