半滑舌鳎（Cynoglossus semilaevis）Nramp基因克隆与表达分析及SNP筛选

摘要

天然抗性相关巨噬细胞蛋白(Natural resistance-associated macrophage protein, Nramp)属于膜整合转运蛋白，具有抑制胞内寄生菌侵染、调节巨噬细胞的抗菌活性等作用。本研究对半滑舌鳎(Cynoglossus semilaevis)Nramp基因进行了克隆和表达分析，并对其与抗鳗弧菌感染相关的单核苷酸多态性(Single Nucleotide Polymorphism, SNP)位点进行了筛选。该基因cDNA序列全长3717 bp，其中开放阅读框(Open reading frame, ORF)1677 bp，所编码蛋白含有558个氨基酸，该蛋白具有Nramp家族的典型特征，包括10个跨膜区(Transmembrane, TM)、1个由20个氨基酸残基组成的胞质内转运蛋白特征结构域(Consensus Transport Motif, CTM)。半滑舌鳎Nramp的ORF末端有1个类似于脊椎动物Nramp2中的铁反应控制蛋白结合位点(Iron-responsive regulatory protein-binding site, IRE)。半滑舌鳎Nramp与其他14个物种的Nramp氨基酸序列同源性在63%−91%之间，系统进化分析表明，半滑舌鳎Nramp和所有鱼类Nramp聚集为一簇，与其他物种Nramp2的亲缘关系较近。实时荧光定量PCR分析显示，Nramp基因在半滑舌鳎脾脏和肾脏中的表达量最高，而在肌肉和性腺中的表达量最低；在哈维氏弧菌感染的半滑舌鳎肾脏、脾脏和肝脏中表达量呈升高趋势，而在鳃中则表现为下调趋势。利用直接测序法检测感染鳗弧菌后同一家系的233个个体(抗病个体165个，感病个体68个)，共检测到15个SNP位点，对其中3个SNP 位点即 SNP-g.3113(T→C)、SNP-g.3125(A→G)和 SNP-g.3164(A→T)进行测序分型后发现，SNP- g.3125(A→G)的等位基因(G)频率和基因型(GG)频率与半滑舌鳎抗鳗弧菌疾病呈极显著相关(P<0.01)。研究结果表明，Nramp 基因不同基因型对半滑舌鳎的抗病能力有着极其重要的影响，SNP-g.3125(A→G)可作为潜在的抗性遗传标记位点。本研究将为半滑舌鳎抗性品系培育提供技术支持。%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 ofNramp gene was amplified from spleen of half smooth tongue sole (Cynoglossus semilaevis) by SMART-RACE. The full-length cDNA ofNrampgene 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. semilaevisNramp 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 ofCsNramp exhibited about 63%−91% homology with 14 other vertebrate Nramp sequences. Phylogenetic analysis revealed that theCsNrampwas clustered with other fish Nrampand was closer to Nramp2 of other species. RT-PCR results of theCsNramptranscripts in different tissues indicated that theCsNramptranscripts were highly abundant in spleen, kidney and low in muscle and gonad. TheC. semilaevis challenged with theVibrio harveyicould evidently elevateNramp 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 inCsNramp gene, this research detected the polymorphisms ofNrampgene in one family of 233 individuals (68 infected individuals and 165 resistant individuals) by direct sequencing. Fifteen SNPs were detected in the partial ofNrampgene 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 differentNramp genotypes, SNP-g.3125(A→G) can be used as potential genetic resistance marker loci, which can provide basic data for the genetic markers ofC. semilaevis resistant breeding.