Cloning and preliminary study on the functions of PmTNFR27 gene in Pinctada fucata martensii

Tumor necrosis factor receptor (TNFR) is an important cytokine receptor, mainly involved in biological processes such as apoptosis, host immune defense, and inflammation. In order to reveal the role of TNFR gene in the immune process of Pinctada fucata martensii, a full length of PmTNFR27 was obtained using rapid amplification of cDNA ends technology, and the expression levels of different tissues, LPS and Poly (I:C) immune stimulation and cadmium stress were analyzed by quantitative real-time PCR (qRT-PCR). Results showed that the total length of cDNA was 1,524 bp, including a 5′UTR of 186 bp, a 3′UTR of 248 bp and an open reading frame (ORF) of 1,062 bp encoding 353 amino acids. Domain prediction showed that PmTNFR27 contained a transmembrane domain and a CRD domain which is typical of the TNFR superfamily. Multiple sequence alignment indicated that PmTNFR27 has low similarity compared with other bivalve species, but its domain regions were highly conservative. Phylogenetic analysis showed that it clustered with other bivalves. In addition, qRT-PCR data indicated that PmTNFR27 was expressed in all tested tissues, with the highest expression in gill. After LPS stimulation, the relative expression of PmTNFR27 in gills reached the maximum at 3 h and decreased to the minimum at 72 h, and the maximum was 9.67 times of the minimum. After Poly (I:C) stimulation, the relative expressin of PmTNFR27 in gills was significantly up-regulated, reached the maximum at 6 h and 12 h, and decreased to the minimum at 96 h, the maximum being13.16 times of the minimum. After cadmium stress, the relative expression of PmTNFR27 reached the maximum at 3 h, and was significantly down-regulated at 24 h and 48 h. This study suggested that PmTNFR27 may be involved in the immune defense response of P. fucata martensii and could provide basis for the further study of the biological functions of TNFR in the shellfish.