导　　师： 王清印;孔杰

学科专业： 070703

授予学位： 博士

作　　者： ;

机构地区： 中国海洋大学

摘　　要： 由白斑综合征病毒/(White Spot Syndrome Virus, WSSV/)引起的对虾白斑综合征病毒病是一种高度传染的病毒性疾病,自1993年爆发以来已经给对虾养殖业造成了巨大的经济损失,但时至今日仍然缺乏有效应对该病毒传播的方法。经过多年的养殖实践和病毒感染实验表明,中国对虾存在一定的抗该病毒感染遗传机制,但具体机制有待进一步阐释。因此,本研究希望从分子水平出发,探讨中国对虾应答WSSV侵染相关的一些基因,为防控WSSV引起的病害提供新的参考资料。本研究以抗病性较强的中国对虾“黄海2号”作为实验材料,对中国对虾应答WSSV侵染相关的基因进行了筛选,并对可能在应答中有重要作用的几丁质结合蛋白/(FcCBP/)、β-1,3-葡聚糖结合蛋白/(FcβGBP-HDL/)、转录抑制子/(FcTR/)、C-型凝集素/(FcCTL/)和谷氨酰胺合成酶/(FcGS/)等五个基因进行了克隆,分析了它们的分子结构特征,预测其可能的生物功能,并研究了其组织分布情况以及WSSV感染后不同组织、不同时间段的表达规律,取得了以下一些结果和结论： 1、通过mRNA差异显示技术/(DDRT-PCR/)分析感染试验中早期濒死和20 d后存活对虾基因表达的差异,共回收46个阳性差异片段。差异片段序列同源性功能分析结果表明,差异片段所编码的蛋白主要参与氧化磷酸化、蛋白质合成、糖酵解、信号转导等生物过程。 2、从中国对虾中克隆了一个新的几丁质结合蛋白/(FcCBP/)基因,全长1250bp。其编码的蛋白质含190氨基酸,在C-末端具有一个2型几丁质结合域/(ChtBD2/),与丽蝇蛹集金小蜂几丁质结合蛋白的相似度最高,为34/%。FcCBP的基因DNA由5个外显子和4个GT-AG型内含子组成。FcCBP在未感染WSSV中国对虾所有检测组织中均有转录,其中肠转录水平最高；感染WSSV后,肝胰腺中的表达在0-6 h迅速上升,然后下降；鳃的表达情况相似,在0-12 h上升至最� White spot syndrome is a highly contagious viral disease caused by white spot syndrome virus /(WSSV/), which resulted in severe economic losses to the shrimp farming industry since its first report in Taiwan in 1993. Until present, there are still no effective ways to cure or completely prevent the prevalence of WSSV. After years of farming practices and WSSV challenge experiments, it showed that there were some anti WSSV infection genetic mechanism, but the specific mechanism needs further explanation. So relevant genes involved in immune response to WSSV challenge in Chinese shrimp Fenneropenaeus chinensis were studied to provide data for the prevention and control of diseases caused by WSSV. In this study, the Chinese shrimp new variety 'Huanghai No.2' which showed better resistance to WSSV in farming practice was used as specific material. Genes involved in immune response to WSSV challenge were screened by DDRT-PCR. The five genes, chitin binding protein /(FcCBP/),β-1,3-glucan binding protein /(FcβGBP-HDL/), transcription repressor /(FcTR/), C-type lectin /(FcCTL/) and glutamine synthetase /(FcGS/) which might play important roles in the response to WSSV challenge were cloned. Their characteristics, possible functions and expression patterns were also studied. The results are summarized as follows: 1. Forty-six positive differential fragments were recovered by DDRT-PCR. Then sequences of the positive differential fragments were determined and blasted in GenBank. Most of them were found to encode proteins related to oxidative phosphorylation, protein synthesis, glycolysis and signal transduction. 2. The FcCBP gene which encoded a protein with a chitin-binding domain type 2 in the C-terminal domain was cloned from F. chinensis by using rapid amplification of cDNA ends /(RACE/). The deduced amino acid sequence of FcCBP showed 34/% identity with Nasonia vitripennis chitin-binding protein. The genomic DN A of FcCBP was composed of five exons and four GT-AG introns. Quantitative realtime PCR analysis r

关 键 词： 中国对虾 一 免疫应答相关基因 克隆

领　　域： [农业科学] [农业科学]