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| Cloning of Tumor Necrosis Factor Receptor Gene(TNFR) and Its Expression in Different Tissues and Response to Vibrio parahaemolyticus Challenge in Large Yellow Croaker(Larimichthys crocea) |
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Abstract The tumor necrosis factor (TNF) and tumor necrosis factor receptor (TNFR) superfamilies play crucial roles in both innate and adaptive immunity. In this study, the fragment of TNFR (LcTNFR) was identified from the EST(expressed sequence tag)database of large yellow croaker (Larimichthys crocea), and the full length cDNA of LcTNFR of 2 016 bp was obtained using SMART-RACE method, consisting of a 5' UTR of 64 bp, an ORF of 1 350 bp and a 3' UTR of 602 bp(GenBank accession No KF432416). The deduced protein was composed of 449 amino acids, with a predicted pI of 5.47 and an estimated molecular mass of 49.7 kD. Protein structure prediction by SWISS-MODEL found that LcTNFR contained 6 α-helix and 0 β-fold. Blast analysis results revealed that LcTNFR had the highest identity with Maylandia zebra 10B-like (74%), followed with Danio rerio ovarian TNFR (51%) and with Carassius auratus TNFR Ⅰ(47%). Phylogenetic tree of the TNFR amino acid sequences analysis furtherly indicated that LcTNFR together with TNFR 10 B - like, TNFR typeⅠ and ovarian TNFR formed a branch. The results of quantitative Real-time PCR (qRT-PCR) showed that LcTNFR was expressed ubiquitously in examined tissues of male and female fishes. The expression level of LcTNFR was significantly higher in ovary than that in other tissues including testis, and the expression level of LcTNFR in spleen, which is an organ involved in immune function, was also higher than that in other examined tissues. In the spleen, the expression level of LcTNFR was up-regulated significantly (P < 0.05) at the 2 day and 4 day after Vibrio parahaemolyticus challenge, and then it restored to the control level at 8 day. The results may provide insight into the immunology research and the development of disease-resistance genetic markers for large yellow croaker.
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Received: 12 September 2013
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