Cloning And Expression Analysis Of Three Immune Genes, And The Primary Methylation Analysis Of CD40 Promoter In Half-smooth Tongue Sole(Cynoglossus Semilaevis)

Half-smooth tongue sole(Cynoglossus semilaevis) is a warm-temperate and large offshore fish which is mainly distributed over the coastal area. As one of the most important economic marine fishes in China, this delicious fish grows rapidly. Along with the enlargement of the cultivation scale, the closed mariculture water continuously deteriorates in recent years. Meanwhile, the bacterial disease confronted by such kind of C. semilavis becomes increasingly serious, which causes mass deaths of C. semilavis farming, seriously restricts the quick development of the industry and leads to enormous economic losses. Thus, it is extremely urgent to study the immune related genes and immune mechanism, so as to promote the long-term health development of C. semilavis farming. As the important members of immune systems, tumor necrosis factor super family and their receptor family play a significant role in the immune response and inflammatory response process.According to the published results of whole genome sequence of C. semilavis, this study cloned three immune related genes including TNF-α1, TNF-α2 and CD40, utilized technique of real-time fluorescent quantitative PCR to detect expression patterns of these three genes in different issues of C. semilavis, and analyzed expression rules in three main immune tissues after Vibrio harveyi infection. Combined with genes sequence and evolvable features, this paper initially revealed their functions in immune response of C. semilavis. Beyond that, bisulfite-sequencing PCR was applied to explore the level of methylation of CD40 gene promoter region in variable mortality families after Vibrio harveyi infection, which is established in 2014. The results are shown below:1. RACE method was applied to clone two homologous genes of tumor necrosis factor, which were respectively named TNF-α1 and TNF-α2. TNF-α1 and TNF-α2 contain 1264 and 1186 nucleotides respectively, and the ORF(open reading frame) were 729 bp and 744 bp, which encoded 242 and 247 amino acids. The putative protein of TNF-α1 and TNF-α2 contains α TNF superfamily motif, a trans-membrane domain, a converting enzyme cleavage site and two conserved cysteine residues. As shown by the alignment result of TNF-α1 and TNF-α2 amino acid sequences of different species, the TNF-α1 and TNF-α2 protein sequence of Cynoglossus semilaevis presents the highest consistency with that of teleostean, which are respectively 38%~61% and 35%~68%. However, the similarity of TNF-α1 and TNF-α2 pellicle precursors was just 32.94%, and the secreted protein similarity was 44.63%. Phylogenetic analysis results displayed that C. semilavis and other teleostean were the first cluster, and other species were the second cluster. Meanwhile, it was distant relative to the TNF-α1 and TNF-α2. According to the real-time quantitative PCR analysis, it shows that TNF-α1 and TNF-α2 were expressed in different tissues of C. semilavis with different expression patterns. The mRNA expression of TNF-α1 gene was relatively high in liver and spleen, yet its expression in other tissues is relatively low. However, the mRNA expression of TNF-α2 gene in liver and kidney was relatively high, while its expression in brain and spleen is relatively low. After infection of Vibrio harveyi, there was significant improvement of TNF-α1 and TNF-α2 expression in three main immune tissues(e.g. liver, spleen and kidney) compared with a PBS control group. In liver, TNF-α1 reached the peak at 12 h, while TNF-α2 achieved it at 6h. In spleen, TNF-α1 had high expression between 6h to 12 h, while TNF-α2 reached the peak at 6h and up-regulated at 24 h. In kidney, TNF-α1 reached peak at 6h, while TNF-α2 achieved it at 24 h. As shown by the above data, TNF-α1 and TNF-α2 were involved in Vibrio harveyi immune responses in C. semilavis.2、According to the results of Whole-genome sequence of C. semilavis, one CD40 homology gene was obtained from the C. semilavis through RACE technology. CD40(Tumor necrosis factor receptor superfamily member 5), as one important receptor molecular, plays a key role in host immune system and takes part in several immune signal pathway. Moreover, the interaction of CD40-CD40 L posed an important impact on the cell signal transduction and activation, host immune response as well as inflammatory response. The full length of the cDNA is 2098 bp, which includes an open reading frame of 1011 bp, a 5′- untranslated region of 44 bp and a 3′- untanslated region of 1043 bp. The ORF of CD40 is encoded 336 amino acids with the molecular weight of 37.27 KD and the Isoelectric Point of 5.355. The 3′- UTR includes two instability motifs(ATTTA) and one polyadenylation signal(ATTAAA) located 28 bp upstream of the polyA tail signal(AATAAA). The putative amino acids contains one signal peptide, one trans-membrane region, two N-glycosylation sites and four conserved cysteine-rich domain. As revealed by amino acid sequence analysis, the C. semilavis CD40 shared 28%-47% certain identity with the other teleost fish, mammal and amphibians, of which the CD40 of C. semilavis had a higher similarity with the bony fish. The results of phylogenetic tree, which corresponds to theamino acid sequence analysis, demonstrated that CD40 of the C. semilavis was clustered in one branch with other teleost fish. As shown by real-time quantitative PCR results, C. semilavis CD40 was the constitutive expression in a wide range of all tested tissues in the healthy fish. Then, after being stimulated by V. harveyiat seven time points(0 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 9 6h), CD40 expression profiles of C. semilavis in three immune tissues(liver, spleen, and kidney) were investigated. The results of the further study revealed that the expression levels were all up-regulated in three immune tissues after stimulation. Moreover, the C. semilavis CD40 expression was highest at 12 h in the liver, ad reached the peak at 6 h in the spleen and kidney. Additionally, the CD40 expression levels of C. semilavis were secondly raised at 48 h in the three tissues. The result of promoter methylation of CD40 gene in liver, spleen and kidney displayed that hypermethylation has a difference in resistance families and sensitive families, meanwhile the hypermethylation of sensitive family was higher than resistance family at the tenth and eleventh GC sites, but the mRNA expressions between the two familes had no difference. The result above suggested that the CD40 played an important role in the host immune system of C. semilavis.