Genomic organization, promoter activity of grass carp MDA5 and the association of its polymorphisms with susceptibility/resistance to grass carp reovirus

Abstract

MDA5 (melanoma-differentiation-associated gene 5), a member of the RLR (retinoic acid-inducible gene I-like receptor) family, plays a pivotal role in innate immunity against viral infection. In the present study, the organization of grass carp (Ctenopharyngodon idella) MDA5 (CiMDA5) gene sequence was elucidated and promoter activity of its 5′-flanking region was detected. In addition, single nucleotide polymorphisms (SNPs) were investigated to explore their association with susceptibility/resistance to grass carp reovirus (GCRV). The CiMDA5 genomic sequence is composed of 10,906bp, containing seven exons and six introns, which is distinct from its counterparts with numbers of exons. The 5′-flanking region of CiMDA5 gene, containing several putative transcription factor binding sites, was amplified using genome walking technique, exhibiting promoter activity. Six SNPs were discovered in the complete sequence of CiMDA5. Five of them locate in introns and one in 5′-flanking region. The genotype and allele distribution were examined by PCR-RFLP in susceptible and resistant fish. The results indicate that genotypes at the −713 C/G and 3338 A/C loci were significantly associated with the resistance to GCRV (P<0.05). Furthermore, at −713 C/G site, no CC genotype individual was found; the mortality in GG genotype group was 42.65%, which was significantly lower than that in CG genotype group (73.91%) (P<0.05). The mortality in CC (33.33%) genotype at 3338 A/C locus was significantly lower than that in AA and AC genotype (61.54%, 45.95% respectively) (P<0.05). The data demonstrate that −713 G and 3338 C carrier are resistant to GCRV. These results provide potential markers for further investigation of selective breeding of resistant grass carp to GCRV.