Correlation Between Nucleotide Polymorphisms Of RIG-I Gene With The Infection Of Grass Carp Reovirus In Grass Carp(Ctenopharyngodon Idella)

Grass carp (Ctenopharyngodon idella), one of the the “Great Four Cultured Fishes” inChina, is favored by fish farmers and customers as its merits of fast growing, low costing andhigh quality meat. However, the growing stocking density is always accompanied by variousdiseases, among which the grass carp hemorrhage disease, caused by grass carp reovirus(GCRV)(a dsRNA virus) wich possesses the most strong toxicit in the aquatic Reoviridaefamily, is the great danger in grass carp cultivationas as its high infectiousness and highpathogenicity. With the havoc of GCRV, there is no effective drug and therapy to prevent it.Recently, the marker assistant breeding based on the immune-related genes is paid muchattention for its potential for the breeding of disease-resistant fish. Retinoic acid induciblegene I protein (RIG-I) is a kind of pattern recognition receptors (PRRs), which can recognizeviral RNA and then activate the signal pathway to induce the expression of type I interferon(IFN) and proinflammatory factors.In order to explore the molecular markers of immune-related genes and geneticcharacters of C. idella RIG-I gene (CiRIG-I) for the future molecular selection of strains thatare resistant to GCRV, DNA was extracted from the collecting samples after a challengeexperiment. Then, the present study investigated the polymorphism sites of CiRIG-I, in which,cloning, sequencing, and genome walking; plasmid construction and cell transfection; DNApooled sequencing and PCR-RFLP were used. In addition, bioinformatic and biometrical toolswere employed for the analysis of genetic information of CiRIG-I and the correlation betweennucleotide polymorphisms and the resistance of C. idella to GCRV infection. At last, thesephenotype-associated polymorphism sites were verified with a second challenge test. Thepresent study obtained following results:1. The full length of RIG-I gene of C. idella (CiRIG-I) is12810bp, containing15exons,14introns and a5’-flanking region with1864nucleotides; and the splicing of intronsaccording with the ‘GT-AG’ principle. 2.5’-flanking sequence of CiRIG-I possesses the promoter activity, which could beenhanced by GCRV inducing.3.19identified polymorphism sites were detected in CiRIG-I, composing by13singlepolymorphisms,3oligonucleotide deletion polymorphisms and3segment deletionpolymorphisms. Among wich,-780C/T and-7402-bp deletion in the5’-flanking region,4731C/T and4945A/G in introns, and254615-bp deletion were significantly associated withthe resistance/susceptibility to GCRV infection.4. Further confirmation proved that the cumulative mortality of-780genotype CC,4731genotype CC and4945genotype AA were significantly lower than that of-780genotypeTT,4731genotype TT and4945genotype GG, respectively; while the deletion mutation at-740and2546sites increased the mortality of grass carp, which may be the hazard of heredityin hemorrhage disease of grass carp.5. Haplotypes the frequencies of3428A-3432G and6610ins-6804ins were significantlylower in resistant stock than that in susceptible stock; while haplotype6610ins-6804del wassignificantly higher in resistant stock than that in susceptible stock.6. All known polymorphisms of CiRIG-I and CiLGP2had no significant interaction.