Molecular cloning and immune responsive expression of MDA5 gene, a pivotal member of the RLR gene family from grass carp Ctenopharyngodon idella

Abstract

The cytoplasmic helicase protein MDA5 (melanoma-differentiation-associated gene-5) recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 5′ triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp Ctenopharyngodon idella. The full length of the C. idella MDA5 ( CiMDA5) cDNA is 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (conserved restriction domain of bacterial type III restriction enzyme), two HELICc (helicase superfamily c-terminal domain) and an RD (regulatory domain). The CiMDA5 mRNA was widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by semi-quantitative RT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P < 0.05), reached the crest at 24 h (7.48 folds, P < 0.05), and then recovered to normal level at 48 h post-injection ( P > 0.05) of grass carp reovirus (GCRV). In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P < 0.05) and returned to control level at 72 h ( P > 0.05) after GCRV challenge. These data implied that CiMDA5 involved in the early stage of antiviral innate immune defense to GCRV in grass carp, and provided new insight into the evolution research of RLR (RIG-I like receptor) gene family.