Complete Mitochondrial Genome, Cloning And Expression Analysis Of CD8in Takifugu Fasciatus

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.