淡水鱼类线粒体DNA D-loop基因的引物设计和应用

Application and Primer Design of Mitochondrial DNA D-loop of Freshwater Fishes

线粒体DNA测序已广泛应用于鉴定和区分种类以及解决系统进化关系问题。本文选取已测定的主要淡水鱼类的线粒体DNA D-loop基因序列进行同源性比较,寻找保守序列,利用简并性原则设计一对通用的简并引物。利用设计的引物对广东省珠江流域主要的淡水鱼类线粒体DNA D-loop控制区基因进行扩增,均能获得单一的目的DNA片断,特异性扩增产物大小为1 kb左右。经测序及与GenBank同源序列的比较,证实为包含线粒体控制区全序列的扩增产物。本研究所设计的引物和应用的方法可以快速地同时对多种鱼类进行大规模的遗传背景分析,鉴定某些难于鉴别的近缘物种,为我国鱼类的种类鉴定、地理种群鉴别及种质资源的评估提供重要的工具。

Mitochondrial DNA （mtDNA） sequencing was widely used to identify, distinguish species and resolve phylogenetic relationships. In this studies, some published mitochondrial DNA D-loop sequences of major freshwater fishes were downloaded and made alignment. A pair of degenerate primers that targeted a portion of transfer RNA （tRNA） -pro and tRNA-phe were designed and used in a polymerse chain reaction （PCR） to amplify the control region. The primers can amplify mtDNA D-loop of 38 freshwater fishes in Zhujiang river area of Guangdong province. The size of amplification product is about 1 kb. It is shown that the primers amplify a D-loop product of correct size only with the mtDNA which was extracted from the species with which the primers were designed to work. This result clearly demonstrates the efficiency and specificity of the primer pairs. The PCR fragments were all cloned and sequenced to confirm that they were indeed D-loop sequences. All mtDNA D-loop sequences were deposited in GenBank. The methods and the primers in this studies are very helpful for large-scale genetic analysis, molecular identification and phylogenetic analysis of freshwater fishes.