家鸽mtDNA ATPase8和ATPase6基因的分子克隆及序列分析

利用特异引物,通过聚合酶链式反应(polymerase chain reaction,PCR)技术,从家鸽(Columbalivia)肝脏组织的总DNA中扩增到目的片段,并将扩增产物克隆到pMD18-T载体中,经菌落PCR与酶切鉴定、序列测定及序列分析.结果表明:克隆得到了家鸽ATPase8-ATPase6基因842 bp及COII的部分序列共861 bp.用DNA分析软件对家鸽ATPase8和ATPase6基因与Genbank中的5种鸟类的ATPase8和ATPase6基因序列进行比较分析,表明家鸽与其他5种鸟类的ATPase8和ATPase6基因具有较高的同源性(88.1%~75.0%),其中与山斑鸠(Streptopelia orientalis)的同源性最高,分别为88.1%和86.5%.家鸽ATPase8和ATPase6基因核苷酸序列的组成中,(A+T)含量分别为55.95%和54.68%,与其它5种鸟类的(A+T)含量(53.5%~60.12%)和(51.9%~54.24%)相近,说明鸟类ATPase8和ATPase6基因序列组成对A+T核苷酸的偏倚程度比较低;而且家鸽该片段的基因组织结构与其他鸟类的基本一致,显示鸟类线粒体基因排列的保守性.家鸽与其他5种鸟类的ATPase8和ATPase6基因序列同源性的分子进化树聚类结果表明家鸽与山斑鸠亲缘关系最近.

Molecular cloning and sequence analyzing of mtDNA ATPase8 and ATPase6 gene in pigeon

Target DNA fragments were amplified by polymerase chain reaction(PCR) from the liver tissue of Columba livia using a pair of specific primers,and the amplified PCR products were cloned into pMD18-T vector.The sequence analyzing revealed that a sequence with a total length of 861 bp including 842 bp nucleotides of ATPase8-ATPase6 gene and partial sequence of the mitochondria CO Ⅱ was cloned.The ATPase8 and ATPase6 genes of Columba livia were proved to have good homology(88.1 %～75.0 %) with the other 5 species of birds recorded in the GenBank and it has 88.1 % and 86.5 % homology with Streptopelia orientalis.The content of A and T(55.95 %,54.68 %) was similar with that of the other 5 species of birds(53.5 %～60.12 %,51.9 %～54.24 %).The pattern of codon usage of the ATPase8 and ATPase6 gene was less biased and the genome structure of the fragment accorded with that other of birds fundamentally.Therefore,mtDNA genes of birds are conservative in arrangement.The phylogenet ic tree based on the nucleotide sequences of the ATPase8 and ATPase6 gene showed that the Columba livia had the most closed genetic relation with the Streptopelia orientalis.