Identification and comparative study of melanoma differentiation-associated gene 5 homologues of triploid hybrid fish and its parents

Abstract

Sterile triploid fish (3n = 150), derived from the hybridization between red crucian carp (Carassius auratus red var., ♀, 2n = 100) and allotetraploid (♂, 4n = 200), exhibits the improved disease resistance compared with its parents, but the current knowledge of the immunity of triploid fish is limited. Here, we report the identification and characterization of melanoma differentiation-associated gene 5 (MDA5) homologues from red crucian carp, triploid fish and allotetraploid. In this study, one red crucian carp MDA5 transcript (2nMDA5), two triploid fish MDA5 transcripts (3nMDA5-a and 3nMDA5-b) and two allotetraploid fish MDA5 transcripts (4nMDA5-a and 4nMDA5-b) have been cloned and identified separately. Immunofluorescence staining assay displayed that these MDA5 proteins were cytoplasmic proteins. RT-qPCR assay showed that, in response to spring viremia of carp virus (SVCV) and poly (I:C) stimuli, the increase of 3nMDA5 mRNA level was obviously higher than those of 2nMDA5 and 4nMDA5. Interestingly, the reporter assay and plaque assay revealed collectively that 3nMDA5-b, a shorter splicing form of MDA5, exhibited the strongest IFN promoter-inducing ability and antiviral activity. Additionally, when co-expressed with 3nMAVS, 3nMDA5-b induced a considerably higher level of IFN promoter activation than 3nMDA5-a; and the interactions between 3nMAVS/3nMDA5-a and 3nMAVS/3nMDA5-b were verified by co-IP assay. Taken together, our findings support the conclusion that in triploid fish, 3nMDA5-b mediates a robust antiviral signaling in host innate immune response.