【作者】 邵爱华；

【导师】 朱江； 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2012， 博士

【摘要】 暗纹东方鲀（Takifugu fasciatus）是具有较高经济价值的鱼类，其产量居鲀形目鱼类之前列，并能进行人工养殖。鲀形目鱼类的分子系统学研究是国内外关注的热点，从种级至目各分类阶元的系统发育研究中，多数是部分或全部用到了线粒体基因组序列。为此，本研究通过设计13对引物进行PCR扩增并测序，首次获得了暗纹东方鲀线粒体基因组DNA (Mitochondrial DNA, mtDNA)全序列，并结合东方鲀属其它种类的mtDNA进行了线粒体全基因组水平的系统发育分析。暗纹东方鲀mtDNA基因组序列全长16444bp，A+T含量为55.8%，其mtDNA由22个tRNA基因、2个rRNA基因、13个蛋白质编码基因和1段819bp非编码的控制区（D-loop）所组成。蛋白质基因除COI和ND6的起始密码子为GTG、CCT以外，均为典型的起始密码子ATG。ND1、ATPase8、COIII、ND4L、 ND5、Cyt b使用典型的终止密码子TAA，其他的使用不完全终止密码子。除ND6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer、tRNAGlu、tRNAPro在L-链上编码之外，其余基因均在H-链编码。基因排列顺序与已测定的鲀类一致。tRNA基因核苷酸长度为64-73nt，预测了22个tRNA基因的二级结构，大多呈较为典型的三叶草状。基于线粒体基因组全序列构建的进化树表明，暗纹东方鲀和红鳍东方鲀(T. rubripes)、中华东方鲀(T. chinensis)聚成一个姊妹群。研究表明，暗纹东方鲀线粒体基因组的组成、顺序、编码链的选择、tRNA的结构以及GC和AT偏斜等，都与典型的脊椎动物线粒体相同，亦未发现重排和假基因。基于东方鲀属鱼类线粒体全序列构建的进化树与形态学分类的阶元关系比较吻合，结果再次支持东方鲀属鱼类为一个单系类群。研究将为暗纹东方鲀线粒体基因组的遗传特点和物种多样性的分析提供分子依据。在暗纹东方鲀的放养过程中，由于集约化养殖的现状会导致鱼体经常受到细菌、寄生虫以及病毒的侵染而导致免疫能力下降。鱼类分子免疫学尚处于起始阶段。在免疫应答研究中，相对于体液免疫相关分子，细胞免疫相关分子的研究更少。因此，本文选取了细胞毒T细胞(CTL)表面辅助受体CD8分子进行研究。CD8是T细胞表面的双链糖蛋白，存在CD8α和CD8β两种亚基形式，是与I型主要组织相容性复合体(MHC I)结合，是T细胞表面受体(TCR)的共受体，也是T淋巴细胞表面的重要标志物，作为粘附分子和辅助受体广泛参与TCR对MHC I分子递呈抗原的识别和信号传递过程。本研究在前期工作基础上，克隆和分析暗纹东方鲀CD8分子两亚基CD8α和CD8β，运用荧光实时定量PCR（Real-time RT-PCR）、免疫组织化学（Immunohistochemistry, IHC）、流式细胞术（flow cytometry, FCM）等方法探讨CD8在mRNA和蛋白质水平上的基因表达特征。克隆获得的暗纹东方鲀CD8α cDNA序列为1533bp，包含一个657bp的开放读码框(ORF)，编码218个氨基酸；其对应的基因组序列为1533bp，包含5个内含子和6个外显子。CD8β cDNA序列长1063bp，ORF长为654bp，编码217个氨基酸，其相应的基因组序列长2639bp，包含6个外显子和6个内含子。生物信息学表明，未成熟的CD8α和CD8β分子多肽序列都由信号肽、胞外可变区、铰链区、跨膜区和胞内区五部分组成。在胞外区可变区和铰链区部分各有两个高度保守的半胱氨酸残基，参与链内和链间二硫键的形成。氨基酸序列多重比对表明，暗纹东方鲀与其他鱼类的CD8蛋白在结构上是相似的，进化上和鲈形目、鲽形目的鱼类关系较近。RT-PCR结果显示，CD8α和CD8β mRNA在暗纹东方鲀组织中的表达量基本上是相一致的，各组织中都有表达但存在差异性。CD8在胸腺中呈现最高的表达水平，在其他组织器官中也有一定量的表达，而在肝脏中的表达量低弱。IHC和FCM研究结果表明，CD8在暗纹东方鲀的组织器官均呈现免疫细胞阳性，但组织间存在一定差异。其中，属于主要免疫器官的头肾、胸腺、脾脏等CD8+细胞数量较多，而属于黏膜样淋巴组织的肠、皮肤、腮等相对于体肾、脑、心脏、肝脏等组织的CD8+细胞检出率较高。这与RT-PCR结果较为一致，提供了有力的形态学和分子水平的依据。这些结果为揭示CD8分子在暗纹东方鲀免疫系统中的作用奠定了基础。更多还原

【Abstract】 Takifugu fasciatus is a kind of farmed fish with high dietary and pharmacological value.Its products are in ahead of the populations of Tetraodontiformes and the fish is easy fordomestic culture.The molecular systematics study is a hot spot in Tetraodontiformes. The part or completemitochondrial genome sequences were usually used to analyze the molecular phylogenetics inTetraodontiformes from species to order levels. In this study, we designed13pairs ofprimers for amplification of the mitochondrial fragments of T. fasciatus by PCR. Aftercloning, sequencing, and overlapping, we obtained the complete mitochondrial genomesequence in the firs time. This sequence was used to analyze the mtDNA genomestructural characteristics and the phylogenetic relationship with some Takifugu species.The sequence of T. fasciatus mitochondrial genome is16444base pairs (bps) inlength with55.8%A+T content, and encodes22tRNAs,2rRNAs,13protein-codinggenes, as well as a control region (D-loop) of819bp. Almost protein-coding genes startwith a typical ATG codon, except for COI and ND6with GTG or CTT. The usual TAAtermination codons were found for ND1, ATPase8, COIII, ND4L, ND5and Cyt b,however, the other7protein-coding genes have an imcomplete termination codons (T orTA). It was comfirmed that the gene arrangement of T. fasciatus could be used toidentify the other Tetrodontiformes fish. Transfer RNA (tRNA) genes range in lengthfrom64to73nt, their planar structures present in characteristic clover leaf.Construction of phylogenetic trees based on the complete mitochondrial genomesequence of Tetrodontiformes species constructed by NJ and MP method. The reslutindicates that the T. fasciatus has closer relationship to T. rubripes and T. chinensis, theyconstitute a sister group. The results also showed that the mtDNA genome of T.fasciatus is similar to most of the vertebrates in gene components, order, orientation,tRNA structures, skews of base GC and AT without rearrangements or pseudogenes.The order relationship of the phylogenetic trees based on complete mitochondrial genome sequence of Takifugu is in accordance with those of the morphologicalclassification. It suggested that the genus Takifugu Abe,1949(Tetrodontiforms,Tetrodontidae) can be treated as a monophyletic group. These data will provide themolecular basis to the genetic characteristics of T. fasciatus mitochondrial genome andthe species diversity.Because of the intensive cultural situation, fish are immunocompromised, andsusceptible to microorganisms, such as bacterium, parasite and virus. Until now lesswere studied in fish immunology, especially in molecular immunology. In the study ofimmune responses, compared with human immunity-related molecules, there are littleknown about cell immunity related molecules. In our study, the cytotoxic T cell (CTL)surface marker, co-recepter CD8, was chosen to analyze. The CD8consists of twodistinguished chains, termed CD8α and CD8β, and functions as a co-receptor for T cellreceptor (TCR) by binding to MHC class I (MHC I) proteins that present peptides onthe cell surface, as well as an important marker of T lymphocytes. It acts as an adhesionmolecule and auxiliary receptor participating in antigen identification and signal transferprocess. Based on previous work, CD8α and CD8β, the two subunits of CD8in T.fasciatus were cloned and their expression levels were detected in mRNA and proteinlevels in different tissues by using Real-time RT-PCR, Immunohistochemistry (IHC)and flow cytometry (FCM).The cDNA of T. fasciatus CD8α was found to be1061bp, with an open readingframe (ORF) of657bp, containing six exons separated by five introns. Itscorresponding genomic sequence was1533bp. The cDNA of T. fasciatus CD8β wasfound to be1063bp, and its ORF was654bp. Its corresponding genomic sequence was2639bp. The CD8β gene consisted of six exons separated by six introns. Both of thepredicted primary structure of CD8α and CD8β contained a signal peptide, anIgV-domain, a hinge region, a transmembrane region and cytoplasmic tail. The twocysteine residues involved in the V-domain of T. fasciatus CD8α and CD8β capable offorming an intra-chain disulphide bond, and the other two cysteines in the hinge regionare conserved in most fish responsible for CD8to form both homodimers andheterodimers. The predicted CD8α and CD8β proteins were structurally similar to those of other fish, especially to those of Perciformes and Pleuronectiformes. The mRNAexpression levels of CD8α and CD8β were basically consistent, but differences exist invarious organs of T. fasciatus. The results of Real-time RT-PCR demonstrated that thehighest expression level of CD8α and CD8β was observed in thymus followed byorgans involved in immunological response, very low expression was detected in theliver. The results of IHC and FCM analysis showed that the ratio of CD8positive cellswas highest in kidney, thymus and spleen, those belong to the major immune organs.While the ratio of CD8positive cells in intestine, skin and gills, which belong to MALT,was higher than that of trunk kidney, heart, brain and liver. These were consistent withthe result of RT-PCR. The CD8was found to be related in T. fasciatus immune.更多还原