Identification And Functional Study Of Seven TRAFs Famaliy Genes In Grouper (Epinephelus Coioides)

Tumor necrosis factor receptor(TNFR)associated factor(TRAF)is a adapter molecule that mediates TNFR,TLR,NLR and RLR signal pathway,and eventually activates NF-?B,MAPK and JNK,and participate in innate and acquired immune responses.Although TRAFs have made great progress in mammal,involving in signal pathway and disease treatment,the function study is still extremely scarce in fish.Cryptocaryon irritans is one of the most common parasite in marine fisheries,which causes great economic losses to aquculture.Studies have shown that fish could defense against the infection of C.irritans by innate immune response and acquired immune response,but the mechanism remain unclear.In this study,the role of TRAFs in C.irritans infection and NF-?B activation were studied,and the results are as follows:Seven TRAF genes,based on the transcriptom database,were cloned from Epinephelus coioides,and respectively named as EcTRAF2-1,EcTRAF2-2,EcTRAF2-3,EcTRAF3,EcTRAF4,EcTRAF5 and EcTRAF7.The open reading frame is1563 bp,1356bp,1797 bp,1767bp,1413 bp,1743bp and 1977 bp,which respectively encod 520 aas,451 aas,598 aas,588 aas,470 aas,580 aas and 658 aas.The results of phylogenetic tree showed that these molecules were homologous to that of mammal and teleost.These TRAFs have a wide distribution in healthy grouper,which could be expressed in 14 tissues.These genes are highly expressed in the mucosal immune tissues,especially in the skin.After C.irritans infection,TRAFs expression were up-regulated in the gills and the head kidney,suggesting that these genes may be involved in the immune responses of E.coioides to C.irritans infection.In HEK293 T cells,with the exception of EcTRAF4,which could be locolized in both cytoplasm and nucleus,other EcTRAFs were locolized in the cytoplasm.In addition,EcTRAF2-1,EcTRAF2-2,EcTRAF3,EcTRAF5 and EcTRAF7 could activate NF-?B,but EcTRAF2-3 and EcTRAF4 did not activate NF-?B.However,when co-transfection with EcTRAF2-1 or EcTRAF2-2,EcTRAF2-3 impaired theactivation of NF-?B induced by both TRAF2 s.Additionally,when TRAF5 lost RING and Zinc Finger domain,the activation of EcTRAF5 was destroyed.