利用PCR-RFLP技术分析拟穴青蟹抗菌肽基因SCY2的SNP位点

利用PCR-RFLP技术分析拟穴青蟹抗菌肽基因SCY2的SNP位点 Analysis of SNP Loci of Antimicrobial Peptide SCY2 Gene in Scylla paramamosain by PCR-RFLP Assay 马洪雨 MA Hongyu1 1 first-author 马春艳 MA Chunyan1 1 张丹 ZHANG Dan1 1 2 崔海玉 CUI Haiyu1 1 2 蒋科技 JIANG Keji1 1 马凌波 MA Lingbo1 1 中国水产科学研究院东海水产研究所/农业部海洋与河口渔业资源及生态重点开放实验室 中国水产科学研究院东海水产研究所/农业部海洋与河口渔业资源及生态重点开放实验室 East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Marine and Estuarine Fisheries Resources and Ecology,Ministry of Agriculture East China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Marine and Estuarine Fisheries Resources and Ecology,Ministry of Agriculture 上海海洋大学水产与生命学院 上海海洋大学水产与生命学院 College of Fisheries and Life Science,Shanghai Ocean University College of Fisheries and Life Science,Shanghai Ocean University 本研究根据公布的锯缘青蟹SCY2基因序列设计引物SCY2-RFLPs和SCY2-RFLPa,利用PCR-RFLP技术分析了5个地理群体（宁德、汕头、万宁、东方、三亚）共计148个拟穴青蟹SCY2基因的多态性.结果表明,所扩增的SCY2基因片段含有2个StuⅠ内切酶识别位点,位于第一外显子的位点没有多态性,位于第一内含子的位点存在多态性.在148个个体中共检测到AA和AB两种基因型,等位基因B的出现频率非常低,只在三亚群体的1个个体中检测到,而在其他4个群体中均未检测到,其等位基因频率为0.003 4.序列分析结果表明,扩增片段长度为1 016 bp,包含部分5′非翻译区（282 bp）、第一外显子（131 bp）和部分第一内含子（603 bp）.AA基因型表现为3条带（从大到小依次为509,356,151 bp）,AB基因型表现为4条带（从大到小依次为660,509,356,151bp）.B等位基因是由于867 bp处发生了C→G的单碱基突变导致StuⅠ内切酶失去了1个识别位点而形成的. The anionic antimicrobial peptide SCY2 gene was recently reported only expressing in gonad tissue of male Scylla serrata.The single nucleotide polymorphisms（SNPs） of functional gene are thought to be potential useful molecular markers for assisting breeding.In the present study,one pair of primers（SCY2-RFLPs and SCY2-RFLPa） was designed according to the gene SCY2 of Scylla serrata firstly,and then the genetic diversity of this gene of five different geographic populations（Ningde,Shantou,Wanning,Dongfang and Sanya） of Scylla paramamosain was studied by PCR-RFLP assay.Two cleavage sites of endonuclease StuⅠ were detected,one of which was showed polymorphism.Two kinds of genotypes of AA and AB were found in 148 individuals,and the frequency of allele B were very low（0.003 4） that was detected only in one individual of Sanya population but none of the other four populations.Sequence analysis showed that the PCR fragment was 1 016 bp in length,with part of 5′UTR（282 bp）,the first exon（131 bp） and part of the first intron（603 bp）.Three bands（509,356,151 bp） were observed from the agarose gels for the genotype AA,and four bands were observed from the agarose gels for the genotype AB.Allele B was associated with the mutation of C→G at 867 bp. 拟穴青蟹 SCY2基因 PCR-RFLP技术 单核苷酸多态性（SNP） Scylla paramamosain SCY2 gene polymerase chain reaction-restriction fragment length polymorphism（PCR-RFLP） single nucleotide polymorphism（SNP） 10.14188/j.1671-8836.2011.04.008 S917.4 国家自然科学基金资助项目（31001106）;上海市科委“科技创新行动计划”基础研究重点科技项目（10JC1418600） 2011-04-01 2021-04-01 4 57