Transcriptome Analysis And IL-12 Immune Response Of Cynoglossus Semilaevis Infected With Vibrio Harveyi

Cynoglossus semilaevis is a marine fish with high economic value.Vibrio harveyi is one of the main pathogens causing bacterial diseases in the process of high-density industrial culture.It has caused serious economic losses to half smooth tongue sole culture and seriously restricted the development of high-density industrial culture of Cynoglossus semilaevis.In this study,the transcriptome of kidney samples from Cynoglossus semilaevis infected with Vibrio harveyi at different time points was sequenced by high-throughput sequencing technique,and the real-time difference of immune response in Cynoglossus semilaevis infected with Vibrio harveyi was explored.Then,the p35 a and p40 c subunits of IL-12 gene were identified and analyzed.The results are as follows.Transcriptome analysis showed that 1297 differentially expressed genes were found in Cynoglossus semilaevis infected with Vibrio harveyi,406 of which were still differentially expressed at 96 h.These genes are mainly involved in nonspecific immunity,including complement,cytokine and its related receptors.In GO(gene ontology)and KEGG(Kyoto Encyclopedia of genes and genome)enrichment analysis,differential genes were mainly enriched in 43 signal pathways,mainly in signal transduction,immune system,signal molecules and interaction pathways.The IL-12 gene of Cynoglossus semilaevis was selected for further study combined with the important cytokines in the signal pathway enriched by transcriptome data.One subtype of IL-12 was identified in Cynoglossus semilaevis.IL-12 is composed of p35 and P40 subunits.The length of coding region is 651 BP and 984 BP,respectively,encoding 216 and 327 amino acids.The predicted molecular weight of IL-12 is 24.11 k Da and 37.78 k Da,respectively,and the theoretical isoelectric point is 7.49 and 6.57,respectively.Phylogenetic tree analysis showed that the identified p35 subunit of Cynoglossus semilaevis belonged to p35 a subunit of fish,and the identified p40 subunit of Cynoglossus semilaevis belonged to p40 c subunit of fish.Amino acid homology analysis showed that the p35 a subunit of Cynoglossus semilaevis had the highest similarity with Takifugu rubripes,and the p40 c subunit of half smooth tongue sole had the highest similarity with Gasterosteus aculeatus,52.04% and 48.67% respectively.The results of homologous modeling of the protein tertiary structure of p35 a and p40 c in Cynoglossus semilaevis showed that p35 a protein of Cynoglossus semilaevis contained more α helices,which was a typical four helix topology of p35 subunit;p40c protein of Cynoglossus semilaevis contained more β folds;p35a protein and p40 c protein covalently combined to form heterodimer in a way similar to ligand and receptor binding.Tissue expression analysis showed that p35 a was highly expressed in gill,brain,heart and ovary,and p40 c was highly expressed in liver,spleen,gill and heart.The expression patterns of p35 a,p40c and their related genes(ifn-γ,IL-10)in Cynoglossus semilaevis infected by Vibrio harveyi were analyzed.The expression of p35 a in spleen increased significantly at 48 h(p < 0.05),and then decreased gradually.In liver and intestine,the expression increased significantly at 72 h.P40 c expression in spleen and liver increased significantly after 6 h;In kidney,it was up-regulated at 48 h;In intestine,it began to increase at6 h,and rose to the highest point at 48 h.These results indicate that the p35 a and p40 c subunits of IL-12 respond to the infection of V.harveyi.The expression of ifn-γ increased significantly in liver and intestine before p35 a,and later in spleen than p35a;the expression trend of IL-10 in liver,spleen,kidney and intestine was opposite to that of p40 c.After overexpression of p35 a and p40 c genes in lymphocytes of Cynoglossus semilaevis,the expression of ifn-γ,tnf-α,tnf-β and other immune related cytokines were detected after stimulation with different concentrations of lipopolysaccharide(LPS).It was found that overexpression of p35 a and p40 c genes in Cynoglossus semilaevis significantly increased the expression of tnf-α under LPS stimulation.