暗纹东方鲀线粒体细胞色素b及其侧翼tRNA基因的克隆与序列分析

以暗纹东方纯（Takifugu fasciatus）肝脏的线粒体DNA为模板，按照红鳍东方豚（Takifugu rubripes）线粒体DNA序列设计合成特异引物进行PCR扩增，克隆并测定线粒体细胞色素b（Cyt b）及其侧翼tRNA基因的全序列，结果显示，克隆的暗纹东方纯Cyt b基因（1137bp）及其5’端上游的tRNA^Glu基因和3’端下游的tRNA^Thr基因序列共1327 bp。用DNA分析软件对暗纹东方纯与GenBank中10个目13种鱼类的Cyt b序列进行比较分析，显示暗纹东方纯与这些鱼类的Cyt b基因具有较高的同源性，与同属红鳍东方纯的同源性最高，为96．1％；与同目不同科的矛尾翻车纯（Masturus lanceolatus）和翻车纯（Mola mola）的同源性分别为74．1％和74．8％。tRNA^Glu基因由69个碱基组成，tRNA^Thr基因由72个碱基组成，与红鳍东方纯相应tRNA的碱基组成完全相同。推定的这两种tRNA的二级结构都具有典型的三叶草型结构，各臂的碱基配对率高，稳定性好。根据暗纹东方纯与其他13种鱼的Cyt b基因序列同源性所建立的进化树比较，结果与传统的分类地位基本吻合。

Cloning and analysis of sequences of Cyt b and its connected tRNA genes of mitochondrial genome from Takifugu fasciatus

Mitochondrial DNA(mtDNA) of Takifugu fasciatus was prepared as templates from the liver tissue.The Takifugu fasciatus mitochondrial cytochrome b(Cyt b) and its connected tRNA genes were amplified by PCR with a pair of specific primers which were designed based on Takifugu rubripes from GenBank.A sequence with total length of 1?327?bp including 1?137 nucleotides of Cyt b gene and partial sequences at both sides was cloned.The Cyt b gene of Takifugu fasciatus was proved to have good homology with other 13 species of fishes recorded in GenBank and it has 96.1% homology with Takifugu rubripes,about 75% with Mola mola and Masturus lanceolatus.The tRNA~(Glu) gene has 69 nucleotides and the tRNA~(Thr) has 72 nucleotides. Their sequences are the same as those of Takifugu rubripes.Proposed secondary structures for tRNA~(Glu) and tRNA~(Thr) genes suggest that they have classical cloverleaf structures with high complementary base rate of each arm and the structures have good stability.The phylogenetic tree shows that the phylogenetic relationships based on the homology of cyt b genes of Takifugu fasciatus and the other 13 species of fishes are consistent with the morphological taxonomic results.