鲤鱼感染嗜水气单孢菌后脾脏转录组的比较分析及免疫相关基因SCARA的系统研究

发布时间：2018-09-01 10:01

【摘要】：鲤鱼是我国重要的经济鱼类,同时也是世界上重要的淡水养殖和观赏鱼类,其在世界上的养殖范围超过了100个国家,占到世界10%的淡水水产养殖产品份额。嗜水气单胞菌广泛分布于池塘,江河,湖泊等环境中,是一种典型的人-畜-鱼共患病病原菌,可以侵染脊椎动物,包括人类,爬行动物以及鱼类,引起败血症等症状。在鲤鱼养殖过程中,经常有感染嗜水气单孢菌并导致大量死亡的情况发生,为了更深一步探究鲤鱼感染病原菌过程中相关免疫机制的应答情况,我们利用高通量测序技术对鲤鱼感染嗜水气单胞菌后三个不同时期脾脏的转录组进行了测定,一共得到了545,000,000个125bp读长的序列,每一个文库都至少得到了两千三百万个序列,所有的原始数据在去除掉模糊的、低质量(Q20)和过短的(长度小于20bp)核酸序列后,得到536,000,000个高质量的序列。我们采用Cufflinks和Tophat等生物信息学软件对所得数据进行分析,在三个不同的时期(感染后4小时,12小时和24小时)共鉴定出2900个差异表达的基因,其中在4小时处显著上调和显著下调的基因个数分别为739和248,而在12小时处显著上调和显著下调的基因个数分别为953和481,在24小时处显著上调和显著下调的基因个数分别为941和704,在三个时间点共同显著上调和显著下调的基因个数分别为243和18。我们随机选取了23个差异表达的基因进行荧光定量pcr验证,结果发现通过转录组分析得到的数据与定量pcr试验得到的差异基因的表达情况基本一致,这也说明了我们转录组分析结果的可靠性。通过对这些差异基因进行go富集分析,我们共获得29个显著富集的go类别,其中包括免疫反应,外部刺激反应,信号转导活性,辅因子结合和应激反应等多种与机体免疫相关的类别,同时根据通路分析和相关基因功能注释信息,我们筛选了一些在信号通路上非常重要的基因,这些基因极有可能参与了鲤鱼应对嗜水气单胞菌的免疫反应,我们对部分基因进行了逐一讨论分析。为了在基因层面更加细致的研究免疫相关基因在鲤鱼中的免疫机制和表达情况,我们选择了a型清道夫受体基因家族并对其序列结构,系统进化,在健康鲤鱼多个组织中和早期发育时期的表达情况进行了分析。我们在鲤鱼的基因组资源中一共鉴定出7条a型清道夫受体基因,分别是scara2-1,scara2-2,scara3-1,scara3-2,scara4-1,scara4-2和scara5,通过对其进行线性分析和系统分化的研究我们发现部分鲤鱼a型清道夫受体基因可能出现基因复制和丢失现象;通过探究鲤鱼a型清道夫受体基因在不同发育时期的表达情况我们发现其广泛存在于鲤鱼的早期发育过程中,且同源基因的两个拷贝之间在不同的发育阶段存在着相似表达谱。我们发现a型清道夫受体基因广泛分布于鲤鱼的九个健康组织中,但是又呈现一定的组织特异性。为了探究鲤鱼a型清道夫受体基因在受细菌感染后的表达情况,我们选取免疫组织脾脏和粘膜组织肠道来进行更深一步的研究。我们发现肠道中A型清道夫受体基因在感染嗜水气单胞菌后4小时就立即出现了显著高表达,而在脾脏中这一情况则发生在12小时处,我们推测这可能与肠组织的免疫识别和脾脏组织的免疫清除有关。本文期望通过整体的转录组表达和相关免疫基因的分析能有助于我们更好的理解鱼体的在受病菌感染时的相关免疫机制,为更进一步的分子辅助育种提供帮助。
[Abstract]:Cyprinus carpio is an important economic fish in China, and it is also an important freshwater aquaculture and ornamental fish in the world. It is cultured in more than 100 countries, accounting for 10% of the world's freshwater aquaculture products. Pathogens can infect vertebrates, including humans, reptiles and fish, causing septicemia and other symptoms. In carp culture, there are often cases of infection with hydrophilic monospora and leading to a large number of deaths. In order to further explore the immune response to the pathogenic bacteria in carp, we use high A total of 545,000,000 125 BP readout sequences were obtained from the spleens of carp infected with Aeromonas hydrophila at three different stages. Each library had at least 23 million sequences. All the original data were removed from the ambiguous, low-quality (Q20) and short (length less than 20b). We used Cufflinks and Tophat bioinformatics software to analyze the data. A total of 2 900 differentially expressed genes were identified at three different stages (4 hours, 12 hours and 24 hours after infection), of which 2 900 were significantly up-regulated and down-regulated at 4 hours. The number of genes significantly up-regulated and down-regulated at 12 hours were 953 and 481, respectively. The number of genes significantly up-regulated and down-regulated at 24 hours were 941 and 704, respectively. The number of genes significantly up-regulated and down-regulated at three time points were 243 and 18, respectively. The results of fluorescence quantitative PCR analysis showed that the expression of differentially expressed genes obtained by transcriptome analysis was basically consistent with that obtained by quantitative PCR test, which also showed the reliability of our transcriptome analysis results. In addition, many immune-related classes, including immune response, external stimulus response, signal transduction activity, cofactor binding and stress response, have been identified. Based on pathway analysis and functional annotation of related genes, we have screened some genes that are very important in signaling pathways. These genes are most likely involved in carp response. In order to study the immune mechanism and expression of immune-related genes in carp more carefully at the gene level, we chose a scavenger receptor gene family and its sequence structure, phylogenetic evolution, in many tissues of healthy carp. We identified seven scavenger receptor genes, SCARA 2-1, SCARA 2-2, SCARA 3-1, SCARA 3-2, SCARA 4-1, SCARA 4-2, SCARA 4-1, SCARA 4-2 and SCARA 5, respectively, from the genomic resources of common carp. Receptor genes may be duplicated and lost. By investigating the expression of scavenger receptor type a gene in carp at different developmental stages, we found that it was widespread in the early developmental stages of carp. Similar expression profiles existed between two copies of homologous genes at different developmental stages. In order to investigate the expression of carp scavenger a receptor gene after bacterial infection, we selected immune tissues from spleen and mucosal tissues from the intestine for further study. We speculate that this may be related to intestinal immune recognition and immune clearance of splenic tissues. We hope that the whole transcriptome expression and the analysis of related immune genes can be used in this study. It is helpful for us to better understand the immune mechanism of fish infected by pathogens and provide help for further molecular assisted breeding.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S943

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