Abstract:Natural resistance-associated macrophage protein (Nramp) belongs to the integration of membrane transport proteins, which has the capacity of enhancing macrophages that are meant to kill pathogens and innate resistance to intracellular parasites. In present study, cDNA of Nramp gene was amplified from spleen of half smooth tongue sole (Cynoglossus semilaevis) by SMART-RACE. The full-length cDNA of Nramp gene was 3717 bp, including 1677 bp open reading frame (ORF) encoding a protein with 558 amino acid residues, which contained the signature features of the Nramp protein family: 10 transmembrane (TM) domains, a consensus transport motif (CTM) with 20 amino acid residues. Compared with the other fish’s Nramp, C. semilaevis Nramp was the presence of one iron-responsive regulatory (IRE) protein-binding site in the terminal of ORF, which was similar to the vertebrate Nramp2. The deduced amino acid sequence of CsNramp exhibited about 63%−91% homology with 14 other vertebrate Nramp sequences. Phylogenetic analysis revealed that the CsNramp was clustered with other fish Nramp and was closer to Nramp2 of other species. RT-PCR results of the CsNramp transcripts in different tissues indicated that the CsNramp transcripts were highly abundant in spleen, kidney and low in muscle and gonad. The C. semilaevis challenged with the Vibrio harveyi could evidently elevate Nramp mRNA levels in spleen, kidney and liver, but the opposite phenomena were observed in the gills. To explore genetic variation and its relevant molecular markers in CsNramp gene, this research detected the polymorphisms of Nramp gene in one family of 233 individuals (68 infected individuals and 165 resistant individuals) by direct sequencing. Fifteen SNPs were detected in the partial of Nramp gene and 3 of them were genotyped successfully and SNP-g.3125(A→G) was significantly correlated to the resistance to Vibrio anguillarum. The results indicated that there were important effects on disease resistance of different Nramp genotypes, SNP-g.3125(A→G) can be used as potential genetic resistance marker loci, which can provide basic data for the genetic markers of C. semilaevis resistant breeding.
