Identification Of Tumor Necrosis Factor Receptor Associated Factor (TRAF) Family And PKC-alpha Genes And Their Role In Immune Response Of Cynoglossus Semilaevis

The tumor necrosis factor(TNF)receptor associated factors(TRAFs)possessed crucial role as the signaling mediators for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor(IL-1R/TLR)superfamily.TRAFs collectively play important functions in multiple biological process and organism immunity.However,the systematical identity of the TRAF gene family in teleost fish haven't been reported,few information was available about their role in innate immunity in Chinese tongue sole(Cynoglossus semilaevis),a high ecomomic value of aquaculture fish.In the present study,bioinformatic analysis identified seven TRAF genes,Cs TRAF2 a,Cs TRAF2 b,Cs TRAF3,Cs TRAF4 a,Cs TRAF5,Cs TRAF6 and Cs TRAF7 in Chinese tongue sole,and the complete ORF sequences of Cs TRAF2 a,Cs TRAF2 b,Cs TRAF3,Cs TRAF4 a,Cs TRAF5 and Cs TRAF7 were cloned,the full-length of the ORF regions of the seven TRAF genes(Cs TRAF2a-Cs TRAF7)were 1791,1629,1779,1413,1668,1731,1965 bp,respectively.A sequence analysis revealed that the genomic structures of Cs TRAFs were various,and contains typical conserved domains compared with mammalian TRAFs.A phylogenetic analysis indicated that the evolutionarily relationships of TRAF family members in teleost fish,and the absence of TRAF1 in most species and TRAF5 in some species of teleosts.Analysis of the gene structure and motifs showed that diversity and distribution of exon-intron structure and conserved motifs in Chinese tongue sole and several other teleost species.Real-time quantitative PCR was used to detect the expression patterns of Cs TRAF genes in the healthy tissues,and in the gill,liver and spleen after bacterial infection with V.harveyi.The results indicate that only Cs TRAF2 a is relatively highly expressed in the brain,and the other Cs TRAFs are highly expressed in immune-related tissues and participate in the immune response after infection with pathogenic bacteria.The relative expression levels of most Cs TRAF genes(Cs TRAF2 b,Cs TRAF3,Cs TRAF4 a,Cs TRAF5,Cs TRAF6)were significantly up-regulated after V.harveyi challenge.Only CsTRAF7 exhibited down-regulated first,followed by a significantly increased,whereas not significantly beyond the original level in all examined three important immune-related tissues(gill,liver,spleen)post-infection.Functional verification of Cs TRAF3,Cs TRAF4 a and Cs TRAF6 revealed that only Cs TRAF6 can activates NF-?B after over-expression of Cs TRAF3,Cs TRAF4 a or Cs TRAF6 in HEK-293 T cells.In addition,the mi RNA mi R-33-p5 targeting the regulation of the TRAF family member Cs TRAF6 was preliminarily verified.This systematic analysis provided valuable information for diverse roles of Cs TRAFs in the immune response.Protein kinase C(PKC)plays an important role in T and B lymphocyte mediateds the immune response.However,the function of PKC is rarely reported in teleost fish.In this study,we have cloned the PKC? gene(namely Cs PKC?),a typical member of the PKC family,and analyzed its molecular characterization feature and expression patterns.Cs PKC? c DNA is 2315 bp,with an open reading frame(ORF)of 2013 bp.q RT-PCR analysis showed that Cs PKC? is widely expressed in various tissues and has a high transcription level in gill,liver,skin and intestine,suggesting that it may play a role in immunity.Subsequently,we have examined the expression levels of Cs PKC? at different time points in six immune-related tissues(gill,liver,skin,intestine,spleen and kidney)after infection with Vibrio harveyi infection.It was found that Cs PKC? is signicantly up-regulated in gill,kidney,skin and spleen and down-regulated in the liver after infection.The results suggest that Cs PKC? might be involved in the immune response to V.harveyi,whether it is involved in macrophage activation in bacterial pathogen infection and the signaling pathways associated with T and B cells still requires further investigation.