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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。
Genomic DNA was extracted from the muscle tissues of four species of marine fish such as Asclepiatus constricta, Scophthalmus maximus, Turbot and Pagrus major by protease k phenol-chloroform extraction. According to the mtDNA (m tDNA) fragment was designed to amplify the mtDNA fragment of about 9,400 bp from 16SrRNA to ND4. The amplified m tDNA was digested with 10 restriction enzymes (H inf I, H ind III, EcoR V, EcoT22 I, Mun I, EcoT14 I, Apa I, B ln I, Ava II, D ra I) Long fragments were restriction digested and analyzed, of which 8 enzymes have restriction sites. The fragment sharing degree of haplotype was calculated by NeiLi fragment method, the genetic distance of four species of fish was calculated according to the fragment sharing degree, NJ phylogenetic tree was constructed by M ega 3.1 software, and the mitochondrial PCR- RFLP method to study the characteristics of fish system.