家蚕地方品种线粒体基因组A+T丰富区的序列及分子进化分析

Nucleotide Sequences in the A+T-rich Regions of Mitochondrial DNA From Local Races of Bombyx mori and Their Molecular Evolution

为了研究家蚕线粒体基因组A＋T丰富区的结构和家蚕品种的进化，用PCR方法扩增了12个家蚕（Bombyx mori）地方品种线粒体基因组A＋T丰富区及其侧翼序列，分离纯化后克隆到pMD18-T载体进行测序。序列分析表明，克隆片段长度约1．1kb，基因排列顺序与C108线粒体相同，依次为12SrRNA基因3’端、A＋T丰富区、tRNA^Met、tRNA^lle。tRNA^Gln和ND2基因5’端，在tRNA^Gln和ND2基因之间有47bp的非编码区。以日本野桑蚕（Bombyx mandarina）为外群，用Phylip软件包构建了基于12个家蚕品种线粒体基因组A＋T丰富区序列的NJ进化树。结果显示，甘肃种单独聚为一群，其进化早于其它11个品种聚成的类群，说明甘肃种是供试家蚕品种中进化最早的品种。这一结果在分子水平上为黄河流域是家蚕品种的发祥地之一提供了证据，也进一步支持了家蚕品种的中国起源说。对A＋T丰富区及其侧翼基因的结构分析表明，家蚕线粒体12SrRNA和ND2基因都十分保守，14个品种统计只分别发生1个和2个碱基转换；A＋T丰富区中（A＋T）比例高达94．9％以上，第27nt开始有1个T-串结构，长度为16～19bp不等；同时还根据3个tRNA基因的核苷酸序列推定了其二级结构。

To study the structure of A ＋T-rich region of mitochondrial DNA （mtDNA） of silkworm, Bombyx mori, and evolution of silkworm race, the A ＋T-rich regions of mtDNA from 12 local silkworm races were amplified by polymerase chain reaction （PCR） method and cloned into pMD18-T vector after purification for sequencing, Sequence analysis revealed that the cloned fragments were about 1.1 kb in length with the same gene order as C108 mtDNA. 3＇-end portion of 12S rRNA, A ＋T-rich region, tRNA^Met, tRNA^lle, tRNA^Gln and 5＇- end portion of ND2 successively. Between tRNAG＇n and ND2 there was a 47 bp non-coding region. Molecular phylogenetic analysis was carried out and a N-J tree was constructed based on the nucleotide sequences of A ＋ T-rich regions of mtDNA from 12 local silkworm races with Phylip software package. The results showed that Gansu race formed one group by itself and its evolution was earlier than the other group formed by the other races, strongly suggesting that Gansu race is the earliest evolved Bombyx mori race in the study. And it did therefore not only provide an evidence in the molecular level for the theory that the Yellow River valley was at least one of the origin of Bombyx mori, but also support the theory of China origination. The 12S rRNA and ND2 genes of B. mori mtDNA are highly conservative and only one and two base substitutions were occurred, respectively, in the 14 races examined. In the A ＋T-rich region, the A ＋T- content was extremely high to over 94.9% and there was a T-stretch ranging from 16 to 19 bps starting at the 27 nt. The secondary structures of three tRNAs were deduced based on their nucleotide sequences as well.