摘要
随着聚合酶链式反应(PCR)和DNA测序技术的发展，线粒体DNA(mtDNA)已成为鸟类系统学和进化研究的理想分子标记。由于线粒体假基因与mtDNA具有一定的相似性，经常有研究者用线粒体基因的通用引物从总DNA中扩增出核基因组中的线粒体假基因，即Numts(nuclear mitochondrial DNA segments，简称Numts)。如果在系统发育研究中将Numts误认为是mtDNA，就很有可能会得出不准确或错误的结论。虽然Numts序列在一定程度上给mtDNA的应用带来了干扰，然而Numts序列是联系细胞核内外两套遗传物质的重要桥梁，另外Numts序列作为核基因组中不表达的假基因也为系统发育研究提供了新的分子标记，为从一个全新的角度来探讨物种的进化机制提供了新的线索。目前人们已利用Numts作为系统发育标记对人和A．thaliana进行了探索性研究，然而对于鸟类核基因组中的线粒体假基因研究却很少。线粒体假基因已成为一个新的热点研究领域，对鸟类Numts进行深入研究对于解决鸟类系统发育中的关键问题具有重要意义。
为了更深入地了解行鸟形目鸟类之间的进化关系以及假基因在分子系统学中的应用价值，本研究首次利用特异性引物采取PCR方法扩增鸻形目11种鸟类的线粒体ATP8／6基因(部分ATP8和部分ATP6基因)及其假基因(Numts)片段。实验中所检测的鸻形目11种鸟类线粒体ATP8／6基因的长度均为626bp，无插入缺失；Numts片段长度均为618bp，与线粒体ATPS／6基因相比存在一处长为8bp的缺失，未见插入现象，并且在序列内部含有终止密码子。鸻形目11种鸟类Numts的核苷酸变异值从0.2％到1.6％，共有14个可变核苷酸位点，转换颠换的比值为1.2，碱基替换在各密码子上发生的频率几乎相同(分别为34.6％，32.3％，33.1％)，没有明显的转换倾向性；而线粒体基因的核苷酸变异值从8.7％到25.8％，共有243个可变核苷酸位点，转换颠换的比值为1.6，碱基替换在密码子三个位点发生的频率分别为20.08％，4.6％，74.6％。用线粒体ATP8／6基因和Numts序列分别构建NJ树和MP树，系统发育分析显示线粒体基因能够很好的反映
With the concurrent developments of polymerase chain reaction (PCR) and DNA sequencing technology, the mitochondrial DNA (mtDNA) has become a very useful molecular marker for studying avian systematics and evolutionary biology. Since Du Buy and Riley firstly found that the mtDNA copies might exist in nuclear genome, many research results have demonstrated that the mitochondrial pseudogenes have been integrated into the nuclear genomes. Especially researchers, in the last ten years, have amplified many nuclear mitochondrial DNA segments (Numts) from total DNA using universal primers. Numts, however, may also embarrass the applications of mtDNA. The previously study displayed that people have obtained inaccurate or false conclusions as a result of wrongly using Numts as mtDNA for phylogenetic analyses. But these Numts may also be used as a novel and powerful tool in phylogenetic studies. As a phylogenetic marker, Numts have been applied in the phylogenetic analyses of human, Arabidopsis thaliana. However, in birds, the Numts have only just a few studies as yet. It therefore is very important to further studying the Numts for revealing the molecular evolution of birds.
For further elucidating the molecular phylogeny of Charadriiformes and the utility of Numts in molecular evolution of birds, in this study, we have amplified mitochondrial ATP8/6 genes (partial ATP8/ATP6 gene) and corresponding Numts in 11 species of Charadriiformes. Our findings demonstrated that the length of mitochondrial ATP8/6 genes and corresponding Numts is 626bp, 618bp respectively. By comparing with mitochondrial ATP8/6 genes, these Numts have 8bp deleted and no insertions were found. The percentage of sequence divergence of Numts (variety from 0.2% to 1.6%) is significantly different from mitochondrial ATP8/6 genes (variety from 8.7% to 25.8%), and the variable nucleotide sites (only 14) and ts/tv ratio (1.2.) of Numts also obviously differ from mitochondrial ATP8/6 genes (243,1.6 respectively). In addition, the percentage of variable sites of the first, second and third codon positions of these Numts are 34.6%, 32.3% and 33.1% respectively, without codon position biases. In contrast, these mitochondrial ATP8/6 genes possess clear codon position biases (i.e. 20.8%, 4.6% and 74.6%, respectively). We further reconstructed the Maximum-parsimony (MP) and Neighbor-joining (NJ) trees using mitochondrial ATP8/6 gene and Numts respectively. Phylogenetic analyses indicated that using mitochondrial ATP8/6 genes can uncover clearly
引文
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