暗纹东方线粒体DNA控制区结构和系统发育分析

Characterization and phylogenetic analysis of control region of mitochondrial genome from Takifugu fasciatus

以暗纹东方(Takifugu fasciatus)肝脏线粒体DNA为模板,参照GenBank中红鳍东方(Takifugu rubripes)线粒体DNA序列设计合成特异引物进行PCR扩增,获得了暗纹东方线粒体DNA控制区基因(818 bp)及5′端上游的tR-NAPro基因(71 bp)的全序列。控制区碱基组成为T32.2%,C 19.1%,A35.6%,G13.2%。对照其他已报道的鱼类控制区结构,对暗纹东方控制区的结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB1,CSB2,CSB3)。CSB1、CSB2序列相对保守,TAS与其回文基序可形成稳定的茎环结构,成为H-链复制延伸时的终止识别位点。同时运用DNA分析软件对暗纹东方与GenBank中其他10多种鱼类的mtDNA控制区序列进行比对,并选取东方属的7种鱼类mtDNA控制区序列构建分子系统树。结果显示控制区基因较适于科鱼类中同属不同种的系统发育分析。

Mitochondrial DNA （mtDNA） of Takifugu fasciatus was prepared as templates from their liver tissue. The T. fasciatus control region（D-loop） and its connected tRNA^Pro genes were amplified by PCR amplification with a pair of specific primers which was designed based on T. rubripes from GenBank. A sequence with the complete nucleotide control region and tRNA^Pro gene from T. fasciatus mitochondrial was cloned and directly sequenced. The control region of T. fasciatus was proved to have good homology with other 6 species of Takifugu fish recorded in the GenBank and it has 94.03% homology with T. rubripe, about 92% -94% with other species. The length of this region （D-loop） contains 818 bp nucleotides and the T, C, A and G contents are 32.2 %, 19.1%, 35.6 % and 13.2 % respectively. The mtDNA control region of T. fasciatus could be portioned into three domains, namely, the termination associated sequence domain, the central conserved sequence domain and the conserved sequence block domain：the extended termination associated sequence（ETAS）, three conserved blocks （CSB-F, CSB-E, CSB-D） in the central conserved sequence block domain and three conserved sequence blocks （CSB1, CSB2, CSB3） in the conserved sequence block domain were successfully identified according to their homology to those of other fish. CSB1 and CSB2 are conservative to the same regions of other kinds of fish. TAS and its palidromic motifs can form stable hairpin-loop structure acting as a recognition site for the termination of H-strand elongation. The phylogenetic trees were constructed with MEGA3.0 through neighbor-joining method and maximum parsimony method baed on the nucleotide sequences from Takifugu fish using Tetraodon biocellatus as outgroup. And the results are compared with each other based on the topology and the bootstrap value. It can be concluded that control region can be used for the phylogenetic analysis of the relationships among Takifugu fish. [Journal of Fishery Sciences of China, 2007,14（3）：352- 360]