Screening And Identification Of Disease-resistant Related Single Nucleotide Polymorphism Markers, And Cloning And Preliminary Analysis Of Two Immune-related Genes In Half Smooth Tongue Sole(Cynoglossus Semilaevis)

Half smooth tongue sole(Cynoglossus semilaevis) is one of unique rare mariculture fishes which is deepley loved by consumers for its rich nutritions and delicious taste. The economic value is extremely high and aquaculture development potential is huge. However, the causes of high density and intensive farming mode and environmental pollution lead to bacterial diseases such as ascites, lousy fin, and so on outbreak frequently, restrict the development of Cynoglossus semilaevis factory farming industry severely. Combined traditional selective breeding mode with marker-assisted selection breeding are advantageous to the Cynoglossus semilaevis resistant strain breeding. SNP is considered to be highly application prospect for molecular marker technology. In present study, two immune related genes were cloned, analysed and preliminary researched. In addition, disease-resistant related SNP markers were screened out in Cynoglossus semilaevis. The main results are summarized as follows:1. Cloning and preliminary analysis of immune-related genes of Cynoglossus semilaevisIn present study, two immune-related genes were cloned, structural and expression analysed, then the expression in transcriptional level of Cs Nramp and Cs IL-10 were detected after injected with Vibrio harveyi. What’s more, Cs IL-10 prokaryotic expression was also preliminary studied.1) Cloning and expression analysis of Cs NrampNramp belongs to the integration of membrane transport protein, which has the capacity of enhancing macrophages that are meant to kill pathogens and innate resistance to intracellular parasites. In present study, Nramp gene was cloned from Cynoglossus semilaevis. The full-length Cs Nramp c DNA is 3717 bp with 16 exons, including 1677 bp ORF encoding a protein with 558 amino acid residues, which contained the signature features of the Nramp protein family: 10 TMs domains, a CTM with 20 amino acid residues between the transmembrane domains 6 and 7. Compared with the other fish’s Nramp, Cs Nramp was the presence of one IRE in the terminal of ORF was similar to the vertebrate Nramp2. The deduced amino acid sequence of Cs Nramp exhibited about 63~91% homology with 14 other vertebrate Nramp sequences. Phylogenetic analysis revealed that Cs Nramp was clustered with other fish Nramp and was closer to Nramp2 of other species. RT-PCR results of the Cs Nramp transcripts in different tissues indicated that the Cs Nramp transcripts were highly abundant in spleen, kidney and low in musle and gonad. The Cynoglossus semilaevis challenged with the Vibrio harveyi could evidently elevate Cs Nramp m RNA levels in spleen, kidney and liver, but the opposite phenomena was observed in the gills.2) Cloning and expression analysis of Cs IL-10IL-10 is a multifaceted cytokine that is produced by and effects a variety of cell populations, including NK, macrophages, T and B cells, its main function is to restrain and termination of the inflammatory response. In present study, IL-10 gene was cloned from Cynoglossus semilaevis. The full-length Cs IL-10 c DNA is 935 bp with 5 exons, including 561 bp ORF encoding a protein with 186 amino acid residues with four conservative cysteine(Cys) forming two pairs of disulphide bridges, which contained the signature features of the IL-10 protein family: G-X2-KA-X2-[D,E]-X-D-[ILV]-[FL V]-[FILMV]-X2-[ILMV][EKQR]. The deduced amino acid sequence of Cs IL-10 exhibited about 27~62% homology with 15 other vertebrate IL-10 sequences. Phylogenetic analysis revealed that Cs IL-10 was clustered with other fish IL-10 and was closer to Gallus gallus IL-10. RT-PCR results of the Cs IL-10 transcripts in different tissues indicated that the Cs IL-10 transcripts were highly abundant in liver, heart, skin and intestines. The Cynoglossus semilaevis challenged with the Vibrio harveyi could evidently degrade Cs IL-10 m RNA levels in liver and gills, but the opposite phenomena was observed in the spleen.Expression primers were designed according to the Cs IL-10 c DNA sequence. The recombinant expression plasmid was constructed by p EASY-E1 Expression Kit. The recombinant plasmid was examined by sequencing, and then transformed into the prokaryotic cell E.coli BL21(DE3) for expression under the induction of IPTG. SDS-PAGE tests revealed that the expression products were 21 k D fusion protein in the main form of inclusion. The recombinant fusion protein was effective combined with polyclonal antibody after Western Blot detection.2. Screening disease-resistant related SNP markers of immune relevant genesFor screening disease-resistant related SNP markers of immune relevant genes in Cynoglossus semilaevis population, 233 indivituals were used(68 dead and 165 survival indivituals) in the present study. Firstly, two gene pools were constructed using 20 dead indivituals and 20 survival indivituals, respectively. Secondly, the SNPs screened from the gene pools were verified using 40 indivituals which were used to construct the gene pools for the first time. Lastly, a second verification for 193 indivituals were performed to verify the differences significance of SNPs between dead indivituals and suivival indivituals. The results showed that three SNPs were preliminary screened from Cs TLR-9 and 15 SNPs were screened from Cs Nramp. There is no significant difference was found in Cs TLR-9’s SNPs between dead and survival indivituals. Whereas, significant difference was found in Cs Nramp’s SNP-g.3125 between dead and survival indivituals for the first single individual verification. Then there is significant difference(P ﹤ 0.01) was found in Cs Nramp’s SNP-g.3125 on the allete and genotype for resistance of vibrio anguillarum between dead and survival indivituals for the first second individual verification. As a result, the SNP-g.3125 of Cs Nramp gene can be used as disease-resistant breeding potential genetic marker loci and provide basic research data for Cynoglossus semilaevis resistant strains breeding genetic molecular markers.