| Grass carp (Ctenopharyngodon idella) is regarded as one of most important economicfreshwater species in China for its fast growth, low cost and delicate taste. Its production hasreached the top among the "Great Four Cultured Fish". Because of weak disease resistantability of grass carp, the annual economic losses in aquaculture is great. Nowadays, grass carphemorrhage, caused by Grass Carp Reovirus (GCRV)(dsRNA virus), has become one of themost serious epidemics in juvenile grass carp. Aquatic wokers are looking for the method ofprevetion and treatment for many years, however, the result is not ideal. Thus identifyingpotential genetic markers, which associated with GCRV resiatance, in the innate immunitygenes may shed light on the disease control in aquaculture.Mx protein, induced by interferon, plays impotant antiviral effect and is the hot spot ininnate immunity molecule. MDA5, as a member of the RLR (retinoic acid-inducible geneI-like receptor) family, plays a pivotal role in innate immunity against viral infection.Therefore clarifying the structure of Mx, MDA5gene and screening SNPs associated withdisease resistance is of great value.(1) Polymorphisms in CiMx2and association with the susceptibility/resistance to grasscarp reovirusThere is no available genomic sequence information on CiMx2gene. The CiMx2genomic structure was predicted according to genomic organization of Danio rerio Mxe(ENSDARG00000014427). Based on the available CiMx2cDNA sequence (accession No.AY395698), primers were designed to amplify the genomic sequence gradually. The CiMx2genomic sequence is composed of8108bp, containing12exons and11introns. Camparingdifferent fish Mx genes, the genomic structure is conservative, sharing the same number ofexons. The whole sequence was scanned and five SNPs (7C/T,528C/T,1191C/A,1205G/Aand6477T/G) were found in CiMx2. The association between the SNPs and thesusceptibility/resistance to GCRV was examined by PCR-RFLP. Statistical analyses revealedthat the genotypes1191C/A and1205G/A were significantly associated with grass carpresistance to GCRV (P=0.002,0.003, respectively). The analysis of linkage disequilibrium in paired loci revealed that7C/T and528C/T were highly linkage disequilibrium. However, nosignificant association was found between the haplotype and susceptibility/resistance toGCRV (P>0.05).(2) The association between CiMDA5and the resistance to grass carp reovirusCiMDA5spans10906bp with genomic structure of seven exons and six introns.Camparing different fish MDA5genes, structure in different species vary greatly. The938bp5'-flanking fragment was cloned by genome walking. TFSEARCH on line analysis softwarepredicted some transcription facor binding sites in this region, such as CdxA, HSF, Oct-1,SBF-1, SRY, Dfd, AP-1, c-Ets-. Plasmid pMDA5-EGFP (containg5'-flanking region ofCiMDA5gene) was transfected into CIK cells. After48h, the result revealed that the5'-flanking DNA fragment of CiMDA5gene can drive the expression of EGFP reporter genein CIK cells, thus exhibits promoter activity. By sequencing, six SNPs (-713G/C,2612A/C,3338A/C,3585A/G,4692A/G and9372C/T) were found in CiMDA5. One SNP located inthe5'-flanking region and other five sited in introns. Statistical analyses revealed that-713C/G and3338A/C were significantly associated with grass carp resistance to GCRV.-713C/G SNP was in the5'-flanking region. A putative binding site for the transcription factorc-Ets-was found in G allele; however carrying C allele resulted in abolition of thisrecognition site, which may be one reason for-713G carrier with protection.The results indicated that single nucleotide polymorphism1191C/A,1205G/A in CiMx2and-713C/G,3338A/C in CiMDA5were significantly associated with grass carp resistanceto GCRV. The present study promisingly enlarged potential genes and genetic markers forselective breeding for the GCRV-resistant grass carp in future. |
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