4种海水鱼线粒体DNA大片段的PCR-RFLP分析

用蛋白酶k酚氯抽提法从圆斑星鲽、高眼鲽、大菱鲆和真鲷4种海水鱼的肌肉组织中提取基因组DNA,根据金枪鱼、牙鲆及相关鱼类的线粒体DNA(m tDNA)序列保守片断设计出扩增引物,扩增从16SrRNA到ND4之间约9400 bp的m tDNA长片段。用10种限制性内切酶(H inf I,H ind III,EcoR V,EcoT22 I,Mun I,EcoT14 I,Apa I,B ln I,Ava II,D ra I)对扩增得到的m tDNA长片段进行限制性酶切和分析,其中8种酶有酶切位点。利用Ne i-L i的片断法计算出单倍型的片断共享度,根据片断共享度计算出4种鱼之间的遗传距离,用M ega 3.1软件构建NJ系统关系树,同时探讨利用线粒体大片断PCR-RFLP方法进行鱼类系统发生研究的特点。

Analysis of Long mtDNA Fragments from Four Marine Fishes by PCR-RFLP

Total genomic DNA was extracted from four marine fishes(Verasper variegates,Cleisthenes herzensteini,Turbot Scophthalmus maximus,red seabream Pagrosomus major) using a proteinase K and phenol-chloroform procedure.Long mtDNA fragments were amplified by long and accurate PCR using primers designed based on the mtDNA sequenses of Thunnus thynnus thynnus,flounder Paralichthys olivaceus and other related fishes.Ten restriction endonucleases(Hinf I,Hind III,EcoR V,EcoT22 I,Mun I,EcoT14 I,Apa I,Bln I,Ava II,Dra I) were employed to analyse the amplified products,8 of which produced restriction fragment.Phylogenetic tree was constructed by calculating the genetic distance among the fishes based on the similarity of restriction fragments.The characteristic of PCR-RFLP technique for long fragment of fish mtDNA was also discussed.