巨魾线粒体12S rRNA和16S rRNA基因克隆及多态性分析

Gene Cloning and Polymorphism Analysis of Mitochondrial 12S rRNA and 16S rRNA of Bagarius yarrelli

运用酚-氯仿方法提取12尾采集于云南省境内河口县的巨魾DNA,利用GenBank中鮡科种类设计特异性引物进行PCR扩增和克隆,利用DNAMAN软件进行序列比对以及采用软件MEGA 4.0来分析巨魾的碱基含量、遗传距离和系统进化树.结果显示:(1)得到954bp的12S rRNA基因序列和535bp的16S rRNA基因序列各12条;(2)12尾巨魾的12S rRNA基因、16S rRNA基因以及12S rRNA基因和16S rRNA的合并基因序列的相似性分别为99.89%,99.95%,99.89%;(3)12尾巨魾的12S rRNA,16S rRNA均表现出A+T碱基含量高于G+C碱基含量;(4)12尾巨魾的平均遗传距离为0.002,转换比颠换的平均值为3.065;(5)利用Neighbor-Joining(NJ)法构建的系统进化树表明魾属单独成一支,支持率达到99%,这一结果与传统的形态学分类基本相似.

Twelve Bagarius yarrelli were collected from Hekou County in Yunnan Province,and their DNA was extracted with the phenol-chloroform method.Specific primers were designed according to the conservative region of 12S rRNAand 16S rRNAgene sequences of Sisoridae species deposited in GenBank,DNAMAN software was used to carry out sequence alignment analysis while MEGA 4.0program was used to calculate the base content,genetic distance and phylogenic tree of the fish.The results were as follows.（1） Twelve sequences of 12S rRNA954bp in length and 12 sequences of 16S rRNA535bp long were obtained.（2） Sequence homology between 12S rRNA gene,16S rRNA gene and the total 12S rRNA＋16S rRNA of 12 samples were 99.89%,99.95%,99.89%,respectively.（3） The content of A base of the sequences of 12S rRNA,16S rRNA,and 12S rRNA＋16S rRNAof the 12 B.yarrelli was high.（4） The average genetic distance of the 12 B.yarrelli was 0.002 while their average ratio of conversion and transversion was 3.065.（5） The phylogenetic tree constructed with the Neighbor-Joining（NJ）method demonstrated that B.yarrelli forms a separate category and the approval rating reached99%,which was similar to the traditional morphological classification.