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| 半滑舌鳎(Cynoglossus semilaevis) Nramp基因克隆与表达分析及SNP筛选 |
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邢贺飞1,2,3, 高峰涛1,2,3,4, 张永珍1,2,3, 董忠典1,2,3,4, 陈松林1,2
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1.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;2.青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071;3.上海海洋大学水产与生命学院 上海 201306;4.中国海洋大学海洋生命学院 青岛 266003
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| 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 |
| 关键词: 半滑舌鳎 天然抗性相关巨噬细胞蛋白(Nramp) 基因克隆 RT-PCR 单核苷酸多态性(SNP) |
| DOI：10.11758/yykxjz.20150311001 |
| 分类号: |
| 基金项目:国家自然科学基金项目(31530078)和“山东省泰山学者攀登计划项目”共同资助 |
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| Molecular Cloning, Expression and SNP Screening of Natural Resistance-Associated Macrophage Protein (Nramp) Gene cDNA from Half Smooth Tongue Sole (Cynoglossus semilaevis) |
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XING Hefei1,2,3, GAO Fengtao1,2,4, ZHANG Yongzhen1,2,3, DONG Zhongdian1,2,4, CHEN Songlin1,2
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1.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;2.National Laboratory for Ocean Science and Technology, Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao 266071;3.College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306;4.College of Marine Life Sciences, Ocean University of China, Qingdao 266003
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| 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. |
| Key words: Half smooth tongue sole (Cynoglossus semilaevis) Nramp Gene clone RT-PCR Single Nucleotide Polymorphism (SNP) |