The Cloning And Functional Study Of Tumor Necrosis Factor Receptor And Their Associated Factors Genes Of Pinctada Fucata Martensii

Tumor necrosis factor receptor mediates signal transduction pathways through signaling molecules such as tumor necrosis factor receptor-associated factors involved in host immune defense,inflammation,and environmental stress biological processes.In order to further study its immune function,we analyzed transcriptome to screen out the key tumor necrosis factor receptors and their associated factors genes.We cloned genes,tested its expression patterns in tissues,and its expression patterns after immune stimulation and gene silencing.Finally,we obtained recombinant protein by prokaryotic expression technology.The main results are as follows:(1)Cloning and quantitation of TNFR and TRAF genes: The full-length c DNA sequences of PmTNFR1,PmTNFR2,PmTNFR5(CD40),PmTRAF2 and PmTRAF4 were successfully cloned,and encoded 427,533,527 and 470 amino acids respectively.Sequence analysis revealed that there are two and three TNFR family cysteine-rich region domains,a transmembrane domain,and a death domain in PmTNFR1 and PmTNFR5,respectively;Two TNFR domains and a transmembrane domain exist in PmTNFR2.A RING finger,two zinc finger,one Coiled-coil,one c IAP2 binding site and a TRAF domain in PmTRAF2,and three zinc fingers and a TRAF domain in PmTRAF4.Tissue distribution results showed that the expression of five genes was significantly higher in gill and hepatopancreas than in other tested tissues.(2)Expression profiles of five genes in hemolymph after LPS stimulation and nucleus insertion surgery: After LPS stimulation,the expression levels of PmTNFR1 and PmTNFR5 genes were significantly up-regulated at 48 h,and the expression level of PmTNFR2 gene was increased at 6 h and 48 h.The expression of PmTRAF2 gene was significantly up-regulated at 6 h and 48 h,and PmTRAF4 gene was significantly higher at 6 h.After nucleus insertion,the expression levels of PmTNFR1 and PmTNFR2 genes were significantly up-regulated on the 15 th day,and the expression levels of PmTNFR5 were significantly upregulated on days 5 and 15.The expression levels of PmTRAF2 were significantly upregulated on days 5,15 and 30,There wasno significant difference in expression levels in PmTRAF4.The results showed that five genes were involved in the immune response of P.fucata martensii.(3)Expression analysis of signal molecules from NF-?B signaling pathway after immune stimulatory: We tested the expression profile of LITAF,TRADD,TRAF2,NIK,IKK,and NF-?B after LPS stimulation and nucleus insertion.We know PmTNFR1,PmTNFR2,and PmTNFR5 are significantly up-regulated at 48 h after LPS stimulation and up-regulated on the 15 th day after nucleus insertion.We find that the expression pattern of these signaling molecules were similar to PmTNFR1,PmTNFR2,and PmTNFR5,which indicates that they may involved in immune response by mediating the NF-?B pathway.(4)Signaling pathways were studied using RNAi technology: We tested the expression of PmTNFR1 and PmTNFR5 after they silencing as well as their downstream signaling molecules.The results showed that the expression level of PmTNFR1 was significantly down-regulated on the 4th day,and TRADD,Caspase-8,Caspase-3,TRAF2,NIK,I?? and NF-?B had the same inhibitory expression pattern;PmTNFR5 expression was significant down-regulated on the 1st day,and TRAF2,NIK,IKK and NF-?B had the same inhibitory expression.We speculated that the apoptosis pathway and NF-?B signaling pathway mediated by TNFR1,and NF-?B signaling pathway mediated by TNFR5 participate in the regulation of the immune response.(5)The pET-15b-PmTNFR5 and pET-15b-PmTRAF2 prokaryotic expression vectors were successfully constructed and obtained recombinant proteins through inducing and purifiing.