【作者】 沈玉帮；

【导师】 李家乐； 张俊彬；

【作者基本信息】 上海海洋大学 ， 水产养殖， 2013， 博士

【摘要】 草鱼(Ctenopharyngodon idellus)是我国最主要淡水养殖对象，其产量一直位居我国和世界养殖鱼类第一。在养殖过程中草鱼病害频繁发生，各种传染性疾病的暴发每年都给草鱼养殖业带来了巨大的经济损失。开展草鱼免疫相关基因的功能研究，阐明其与抗病力的相关性，将为草鱼抗病分子育种提供重要的理论基础。本研究拟从克隆获得的草鱼补体Bf/C2、C6和C7基因入手，对其进行结构、连锁和组织表达分析，初步了解其生物学功能，并检测它们内部的基因多态性，进行不同基因型或单倍型与草鱼细菌性败血症的关联分析，以期筛选出有育种指导价值的优势基因型，取得的结果如下：1.草鱼4个补体基因的克隆与表达根据EST拼接成的连接群或斑马鱼的cDNA序列设计引物，从草鱼基因组中克隆到补体Bf/C2A、Bf/C2B、C6、C7基因的部分序列。采用RACE方法获得了4个补体基因的cDNA全长序列，并推导出相应的氨基酸序列，与其它物种的相同基因序列进行了比较分析。这4个基因cDNA序列已提交到GenBank数据库，登录号分别为JF47038，JF47039，HQ416903，JN655169。采用Real-time PCR技术检测了补体Bf/C2A、Bf/C2B、C6、C7基因在健康草鱼成体组织、诱导后不同时点组织和不同发育时期的表达情况。结果显示，补体Bf/C2A、Bf/C2B、C6基因在所检测的12个组织中都有所表达，其中肝和脾中的表达量最高，但C7只有在头肾、中肾、皮肤、脾、心脏和肠中进行了表达。经福尔马林灭活嗜水气单胞菌诱导后，Bf/C2A、Bf/C2B、C6、C7基因在所检测的组织中出现了显著上调。补体C6mRNA在未受精的卵中转录。孵化1d C6mRNA转录显著上升，到第10d又开始下降，这表明补体C6在草鱼胚胎和鱼苗早期起到免疫保护的作用。2.草鱼补体C6、C7基因的连锁分析对补体C6、C7基因在作图家系4个亲本中进行多态性位点筛选，在补体C6基因第4号外显子上获得了1个SNP位点，在补体C7基因第5号内含子上发现了1个SNP位点。将这两个位点在两个作图家系192个子代中进行了基因分型，通过连锁分析发现补体C6、C7基因都位于草鱼第3号连锁群上，而且彼此紧密连锁，分布在微卫星标记CID0050和CID0190之间。3.草鱼补体C6、C7基因的多态性与细菌性败血症的关联分析采用人工感染嗜水气单胞菌的方法，建立了草鱼易感群体(186尾)和抗病群体(191尾)，用于连锁分析。使用基因组步移法获得了补体C6、C7基因的启动子序列，分别为1130bp和1107bp。使用PCR方法获得了补体C6、C7基因组DNA全长分别为9288bp和7068bp，都包括18个外显子和17个内含子。在草鱼C6基因中共发现了9个潜在的SNPs位点，其中编码区的1个同义突变位点，其余8个都分布在内含子上。对其中的8个位点在易感群体和抗病群体中进行了等位基因频率检测，没有发现单个SNP位点与败血症抗性有关联。连锁不平衡分析表明，其中G2638A、G2804C、A3519C、T3591C4个位点之间处于高度连锁状态(r2>80)。单倍型分析显示，共发现了13个单倍型，其中单倍型GCCC与细菌性败血症易感性显著相关(p <0.05，OR=1.45)。在草鱼C7基因中共发现了7个潜在的SNPs位点，其中编码区的1个同义突变位点，启动子区1个突变位点，其余5个都分布在不同内含子上。对其中的6个位点在易感群体和抗病群体中进行了等位基因频率检测，发现SNP位点C1931T与败血症抗性显著相关(p=0.045和p=0.028)。连锁不平衡分析表明，其中A-69C、T819C、A2456T3个位点之间处于高度连锁状态(r2>85)。单倍型分析显示，共发现了8个单倍型，没有发现单倍型与细菌性败血症显著相关(p>0.05)。更多还原

【Abstract】 Grass carp is one of the most impotant fresh aquaculture fishes in China. The yield andoutput value of grass carp are ranked first one of all aquaculture fishes in China and Inthe world. Grass carp is easy to be infected by many pathogens. Various diseases cancause heavy losses in grass carp aquaculture every year. So functional studies on itsimmune factors affecting resistance to diseases were carried out to understand theirrelationship with disease resistance, then will provide the theoretical base for molecularbreeding of grass carp. In this thesis, the grass carp complement Bf/C2, C6and C7genes were cloning and characterized, and the linkage and expression were analyzed tounderstand their biological function. The polymorphisms of complement C6and C7were investigated, and genetype and haplotype were associated with resistance tohemorrhagic disease in grass carp.1. Molecular cloning and expression of four complement gene in grass carpThe primers were designed according to the contigs or zebrafish cDNA sequences. Thepart of the full-length fragments of four grass carp complement genes were isolated, andthe four genes were Bf/C2A、Bf/C2B、C6、C7. RACE method was used to clone four genesin grass carp. The amino acid sequences of the four genes were deduced and comparedwith some of other species close genetically. The full-length cDNA sequences of thisfour genes were submitted to GenBank, and the accession numbers are JF47038,JF47039, HQ416903, JN655169.Real time PCR method was used to investigate the four grass carp genesexpression patterns in various tissues of healthy fish, at different developmental stages,after challenge with Aeromonas hydrophila bacterium. The results show that Bf/C2A,Bf/C2B, C6genes were expressed in a wide range of grass carp tissues, with the highestlevel of expression of these genes detected in the liver and spleen, but C7geneexpression was detected in the trunk kidney, liver, head kidney, skin, spleen, heart and intestine. In response to Aeromonas hydrophila injection, their expressions weresignificantly up-regulated in observed tissues. The expression level of gcC6was high atthe unfertilized egg stage. It was significantly increased at1day post-hatching, but itwas decreased at10days post-hatching. Taken together, our data provided a betterunderstanding of the physiological function of complement C6in vertebrates.2. Linkage analysis of complement C6and C7genes in grass carpFour parental individuals from two families were used to screen the polymorphisms ofcomplement C6and C7genes. A single nucleotide polymorphism loci were found in theforth exon in C6, and another single nucleotide polymorphism loci were obtained in thefifth intron. Finally these two SNPs were used for genetyping the two mapping familiescomprising192individues. The grass carp C6and C7genes were located adjacent toeach other within the chromosome3linkage group between two markers, CID0050andCID0190.3. The polymorphisms of complement C6and C7and their association with resistanceto bacterial hemorrhagic disease in grass carpAfter the challenge with Aeromonas hydrophila, A resource population consisting of186infected individuals and191unfected individuals was constructed. Genomicwalking method was used to obtain the promoter sequences of these two genes, and thesequence length of complement C6and C7were1130bp and1107bp, respectively. Thegenomic sequences of complement C6and C7were9288bp and7068bp, respectively,all consisting of18exons and17introns.Nine SNPs were identified in the genomic sequence of C6. One SNP in exon weresynonymous mutation. Eight of them were sited in introns. We selected the eight SNPsand analyzed the genotype and sllele distribution in susceptible and resistant groups.The statistical results indicated that there was lack of association between C6SNPs anddisease resistance in grass carp. The four SNPs were highly linked each other includingG2638A、G2804C、A3519C、T3591C (r2>80). There were thirteen haplotypes inresource population that generated with the four associated SNPs. haplotype GCCC wassignificantly associated with susceptability to bacterial hemorrhagic disease (p <0.05).Seven SNPs were identified in the genomic sequence of C7. One SNP in exon weresynonymous mutation. One SNP was found in promoter region. Five of them were sited in introns. We selected the six SNPs and analyzed the genotype and sllele distribution insusceptible and resistant groups. The statistical results indicated that C1931T was foundto be associated with disease resistance in grass carp ((p=0.045and p=0.028)). Thethree SNPs were highly linked each other including A-69C、T819C、A2456T (r2>85).There were eight haplotypes in resource population generated with the three SNPs. Nohaplotypes were strongly associated with resistance to hemorrhagic disease (p>0.05).更多还原