野桑蚕线粒体ND5基因及其两端侧翼tRNA基因的克隆与序列分析

Clonging and Squence Analysis of ND5 Gene and its Pericentric Flanking tRNA Genes From Mitochondrial DNA of Bombyx mandarina M.

应用PCR技术,对中国山东青州野桑蚕mtDNA的ND5基因及其两端侧翼区域进行了克隆和序列分析。结果表明,在所测定的2．2kb左右的片段中,含有ND5基因的完整序列及Phe-tRNA(UUC)、Ser-tRNA(AGC)、Glu- tRNA(GAA)、His-tRNA(CAC)等4个tRNA基因,该片段的基因组结构与家蚕及其它地域来源野桑蚕的基因组结构基本一致,显示蚕类线粒体基因排列的保守性。ND5结构基因在不同家蚕及野桑蚕之间的比对分析表明,它们在核苷酸水平、氨基酸水平的同源性很高,相似性达96%以上。4个tRNA基因的碱基错配率较高,TψC环缺乏相对保守的T-ψ-C-Pu-A序列,各臂的碱基配对数、各环碱基数目变化较大。以ND5基因的核苷酸序列及氨基酸残基序列进行了家蚕及野桑蚕的分子进化分析,进一步证明家蚕起源于中国野桑蚕。

The ND5 gene and its pericentric flanking region from mitochondrial DNA （mtDNA） of Bombyx mandarina M. from Qingzhou, Shangdong Province, was cloned and sequenced. The results indicated that the sequenced fragment, about 2.2 kb, contained a intact ND5 gene and 4 tRNA genes i. e. Phe-tRNA （UUC）, Ser-tRNA （AGC）, Glu-tRNA （GAA） and His-tRNA （CAC） genes. And the genome structure of the fragment is in accordance with that of other strains of Bombyx mori L, and Bombyx mandarina M. fundamentally. Therefore, mtDNA genes of silkworms are conservative in arrangement. Further analysis of various Bombyx mori L. and Bombyx mandarina M. showed that their ND5 genes are highly homologous at nucleotide and amino acid level with over 96% identities. But the 4 tRNA genes showed high mismatch rate. Their TψC-Ioops lack the relative conservative sequence of T-ψ-C-Pu-A, and the base pair numbers of each stem and the base number of each loop vary strikingly, Additionally, the molecular evolutionary analysis was carried out based on the sequences of nucleotide and amino acid of ND 5 gene, which furtherly supports the fact that Bombyx mori L. stems from Bombyx mandarina M..