长牡蛎抗病毒天然免疫关键基因的初步研究

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	长牡蛎抗病毒天然免疫关键基因的初步研究
	管旭东
学位类型	硕士
导师	李莉 ; 张国范
	2015-05-23
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物工程
关键词	长牡蛎大规模死亡牡蛎疱疹病毒oshv-1 重测序 Snp Tlr Il17
摘要	牡蛎是世界重要养殖贝类，在我国和世界上很多沿海地区广泛分布。牡蛎养殖产量居海产动物首位，具有很高的经济价值。但牡蛎经常爆发大规模死亡，严重制约着牡蛎养殖业的发展，造成严重经济损失。1972年在美洲牡蛎中首先发现了疱疹样病毒OsHV-1。目前OsHV-1已被证明与牡蛎的大规模死亡有关。但我国鲜有报。对人工培育的长牡蛎幼虫，从担轮幼虫到壳顶幼虫期间定时取样，对累计死亡率高达90%的牡蛎幼虫进行PCR检测，牡蛎幼虫样品OsHV-1病毒阳性率高达98%，并且载毒量较高。表明牡蛎幼虫的大规模死亡与疱疹病毒密切相关。同时对山东几个牡蛎主养海区养殖成体牡蛎OsHV-1病毒感染情况进行了调查。养成期牡蛎死亡个体的病毒阳性率较低（5%-50%），而且与死亡的相关性不大。主要原因是获得的养成期牡蛎死亡样品不是典型的大规模死亡期牡蛎，而且海区的养殖环境较复杂，有多重因素可造成牡蛎死亡。根据长牡蛎对OsHV-1病毒的敏感性存在差异的特点，以人工感染的自然群体牡蛎为研究对象，将病毒感染后死亡的100个个体作为病毒敏感型群体(TM)，将100个病毒感染后存活个体作为病毒抗性群体（TF）。然后分别对两个群体各100只牡蛎DNA进行等量混合，分别构建DNA文库，并使用Illumina二代测序技术对两个群体进行全基因组重测序。利用生物信息学手段将重测序结果与牡蛎参考基因组对比，在长牡蛎全基因组范围内大量发掘基因单核苷酸多态性（SNP）、短插入/缺失（INDELs）等基因多态性变异信息。根据SNP位点差异的方法筛选到的3601个基因中有96个关键病毒候选基因：其中包括10个TLR基因，3个IL17相关基因，3个NF-kB基因等；根据基因Fst结果选取前5%的1401个基因中，有570 个表达谱病毒上调（virus.up）基因。并在此基础上对筛选出的基因进行GO功能富集，结果显示免疫应激功能基因显著富集。鉴于TLR2基因在长牡蛎抗病毒天然免疫通路中的关键作用，我们将TLR2家族四个基因进行区域重测序，并验证其多态性位点。在TLR2-1基因区内编码胞外识别关键结构域LRR-CT的位置发现一个显著差异（p<0.05)的SNP位点，其密码子存在GAA、GAC两种类型，对应的氨基酸为谷氨酸和天冬氨酸。病毒敏感性群体在此位点碱基倾向为C，病毒抗性群体在此位点碱基倾向为A。表明，此位点基因型的变化有可能改变牡蛎的病毒敏感性，这对抗病毒牡蛎分子育种具有重要意义。 IL17是一种重要的促炎性细胞因子。我们利用RACE技术克隆得到两个长牡蛎IL17基因（分别命名为CgIL17-7、CgIL17-8）的cDNA全长。通过与其他物种IL17多序列比对发现二者与其他6个长牡蛎IL17氨基酸序列都含有5个与二硫键形成有关的保守半胱氨酸残基。在进化树分析发现长牡蛎IL17亲缘关系最近，并且与其他无脊椎动物IL17处于同一分支而与脊椎动物分开。利用荧光定量技术对人工感染长牡蛎群体中IL17两个基因的表达分析发现，病毒感染后CgIL17基因在长牡蛎各组织中表达量均有升高，表明CgIL17在长牡蛎病毒免疫应激中起重要作用。总之，本研究对国内长牡蛎大规模死亡状况进行调查，并对长牡蛎抗病毒天然免疫通路关键基因进行筛查、变异位点检测以及表达差异进行分析。本研究将促进OsHV-1病毒引起的大规模死亡机制研究以及病毒抗性育种研究。
其他摘要	Oyster is a worldwide aquacultural species with global distribution, ecological importance, and high economic value. The production of the oyster ranks the first place in mariculture, however the mass mortality of the oyster often occurred all over the world. This restricts the development of the oyster aquaculture and cause economic loss seriously. Oyster Herpesvirus-1 (OsHV-1) was first detected in Crassostrea virginica in 1972. The OsHV-1 has been proved to be relative to the mortality of oyster now. The hatched larvals of the Pacific oyster, C. gigas were sampled at multiple time points from trochophore to umbo larvae and OsHV-1 was detected by PCR in those with the cumulative mortality rates of more than 90%. It showed that the positive rate of the OsHV-1 was 98% and there was a high quantity of OsHV-1 in the C. gigas larvae. The results indicated that mass mortality of C. gigas larvae may be related to OsHV-1. At the same time, mass mortality of grow-out oyster was investigated the areas of Shandong. It was display that less positive rate of the OsHV-1 in death adult oysters (5%-50%), and low correlation between mortality and OsHV-1 were found. The main reason is that oyster samples obtained were not from the typical mass mortality, and the environment factors contributing to the death of the oyster are complex. According to the difference of sensitivity to OsHV-1 in oysters, 100 dead individuals after artificial virus infection were selected as the virus sensitive group (TM), and the other 100 individuals survived as antiviral group (TF). The DNA of 100 individuals of TM and TF were equally mixed respectively. Then whole genome re-sequencing of this two groups were performed using Illumina next-generation sequencing technology. The sequencing reads were aligned to the reference genome sequence. SNPs and short InDels of sequenced genome were detected in the whole genome. 3601 genes were found according to the difference of SNP, among which include 96 (include 3 TLR genes, 3 IL17 and 3 NF-kB) genes were virus-associated identified according to the frequency difference of SNP. In addition, 1401 genes were selected based on the result of genetic Fst (top 5%) which contain 570 up-regulated expression genes of virus-associated expression profiles. The GO enrichment analysis indicated that an enrichment of immune genes. TLR2 genes are important to the antiviral innate immune pathways in the oyster, and SNPs of TLR2 genes were detected by candidate gene re-sequencing. A significant difference SNP (p<0.05) of TLR2-1 was found in the LRR-CT domain, the alternative condons were GAA and GAC, respectively, corresponding to the glutamic acid and aspartic acid. Specifictly, the virus sensitivity group tends to be C in this site, while the antiviralis are inclined to A. It indicated the correlation between genotype of this SNP and the sensitive to OsHV-1, which will contribute to the oyster molecular breeding. IL17 is an important pro-inflammatory cytokine. Two full length cDNA of IL17 genes were cloned by RACE (named CgIL17-7, CgIL17-8 respectively). Multiple alignments indicated that these two IL17 and the other six oyster IL17 amino acid sequences contained five conservative cysteine residues relating to the formation of disulfide bond. The phylogenetic tree showed that 8 oyster IL17s clustered together and all of invertebrates IL17 grouped into a single branch and separated from vertebrates IL17. The expression of two CgIL17 genes was analyzed by qRT-PCR in virus-infected and uninfected samples. The data showed that the expression of CgIL17-7 and CgIL17-8 have increased in virus-infected samples, suggesting the importance of IL-17 in the virus immunological stress In summary, the mass mortality of oyster was investigated, and the mutation and expression of key genes in oyster innate immune pathways were analyzed. The result will help for prevention and treatment of the mortality by OsHV-1 in oyster.
学科领域	海洋生物技术
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22760
专题	海洋生物技术研发中心
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	管旭东. 长牡蛎抗病毒天然免疫关键基因的初步研究[D]. 北京. 中国科学院大学,2015.

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