《岱衢族大黄鱼线粒体基因组全序列的扩增及特异鉴别引物的开发》-毕业论文设计（学术).docVIP

岱衢族大黄鱼线粒体基因组全序列的扩增及其特异鉴别引物的开发 摘要：本研究使用15对引物PCR扩增、克隆获得了岱衢族和闽-粤东族大黄鱼线粒体基因全长序列，结合NCBI上的已知序列FJ 595214和NC011710分析出大黄鱼线粒体基因的高变异区域；通过2对引物扩增了岱衢族和闽-粤东族样本各25条的高变区域，对测序结果分析处理分成3个不同的分类单元（Operational Taxonomic Unit，OTU）：D1-N1、D1-N2和D2；岱衢族大黄鱼25个样品均为D1-N1型；而闽-粤东族大黄鱼中可以分为3个类型：D1-N1型，4个样品，D1-N2型，5个样品，D2型，16个样品；然后设计出能有效区分不同OTU分型的特异鉴别引物J1和J2，并对PCR鉴别的条件进行了优化，退火温度和循环数优选55.8℃、28；使用鉴别引物进行PCR反应电泳拍照后，如果样品分型为D1-N2型和D2型，则100%为闽-粤东族大黄鱼，如果样品OTU分型为D1-N1，则该样品为岱衢族大黄鱼的概率为86.2%，闽-粤东族大黄鱼的概率为13.8%，在此研究基础上，可再结合其它的差异区域分型来更进一步提高岱衢族大黄鱼鉴别的准确率。研究结果为岱衢族大黄鱼的种质资源保护、良种选育和市场销售等方面提供技术支持。 关键词：；岱衢族大黄鱼；闽-粤东族大黄鱼；线粒体基因组；OTU; 鉴别 The amplification of the complete mitochondrial genome of the Dai-qu Stock Pseudosciaena crocea and the development of its diagnostic primers Abstract: The complete mitochondrial DNA(mtDNA) genomes of Pseudosciaena crocea sampled from Min-yue and Dai-qu Stock were amplified by PCR with 15 primer pairs and sequencing, with which high variability regions of Pseudosciaena crocea were analysised combining with known sequences in NCBI (FJ 595214 and NC011710). The sequences of high variability regions from 25 Min-yue and Dai-qu Stock samples respectively were obtained with 2 primer pairs, which were divided into 3 Operational Taxonomic Units (OTUs): D1-N1、D1-N2 and D2. The results indicated that: all the Dai-qu Stock samples were clustered into D1-N1type; Min-yue Stock samples were clustered into D1-N1、D1-N2 and D2 type which accout for 4、5、16, respectively. Primer pairs J1 and J2 were designed to significantly distinguish different OTUs, and PCR conditions were optimizated for identification that annealing temperature and cycle number were 55.8℃ and 28. After PCR procuct amplified with diagnostic primers is photoed by gel imaging system, the accurate rate of identification for Min-yue Stock Pseudosciaena crocea is 100% if the band pattern on photo belongs to D1-N2 or D2 type, and the accurate rate of identification for Dai-qu and Min-yue Stock Pseudosciaena crocea are respectively 86.2% and 1