【摘要】 以中国明对虾WSSV感染6h的头胸部组织为实验材料,提取总RNA分别进行正反向抑制性PCRcDNA消减杂交,消减杂交后的PCR产物经纯化并克隆到T载体,进而转化成正反向消减文库。消减文库库容分别为:正向库,3.7×104;反向库,1.2×104。利用消减文库的克隆构建了cDNA表达谱芯片,共有1536个靶点,其中正、反向文库各768个。使用该cDNA芯片对WSSV感染后6h的对虾组织进行了表达谱分析,对其中80个出现明显表达调控变化的阳性克隆进行了测序。结果显示,在WSSV感染后6h,病毒基因开始大量表达,而宿主糖酵解,嘌呤、嘧啶代谢以及精氨酸代谢等代谢途径的关键基因出现明显下调。这表明病毒已在宿主体内大批量复制并开始抑制宿主代谢。病毒上调表达的WSV482可能与病毒毒力的增强有关,而宿主蛋白的管家基因如Actin,EFα的下调表达提示:在利用基因表达技术研究WSSV病毒的实验中,管家基因的选择要十分慎重。此外,根据作者已有的研究结果,TPI基因是比较好的候选管家基因。而WSV414、WSV215和WSV482是对WSSV进行RNA干涉实验较好的候选靶标基因。更多还原

【Abstract】 After challenged with WSSV for 6h, forward and reverse SSH(suppression subtractive hybridization) were performed by mRNA of cephalothorax of Chinese shrimp, Fenneropenaeus chinensis.The PCR products of SSH were cloned into a T vector, and then transferred into plasmid libraries.The titers of the two libraries were, forward library at 3.7 ×104, and reverse library at 1.2×104.A total of 1536 randomly-selected cDNA clones of the SSH libraries(768 each) were stained on a glass slide to construct cDNA microarray.The gene expression profiles of cephalothorax of the shrimp 6h after WSSV injection were examined in microarray.Eighty significantly differential expression clones were sequenced.The results reviewed that, 6h after WSSV injection, the WSSV genes were expressed at a high mRNA level while the processes of host glycolysis and metabolism of purine, pyrimidine, and arginine kinase were observably down regulated.The results indicated that the up regulation of WSV482 might result in the virulence of WSSV.The down regulation of house keeping genes includes Actin and Efα reminded us that we should carefully choose the inter-stander in such experments.Besides, we believe that TPI is a good candidate of inter-stander based on our previously experiments.WSV414, WSV215 and WSV482 can be good candidates of target of RNA interference.更多还原