12S rRNA、Cyt b基因标记在几类野生动物检材鉴定中的差异

The Differences of 12S rRNA and Cyt b Gene Marker in Several Wildlife Species Identification

【目的】通过比较线粒体DNA上的12S rRNA和Cyt b基因分子标记在几类野生动物物种鉴定中的效果差异,以确定基因标记技术在野生动物物种鉴定中的有效性。【方法】提取各类野生动物样本DNA,PCR扩增线粒体DNA上的12S rRNA基因片段和Cyt b基因片段,对PCR产物进行电泳检测及测序分析,测序结果在Gen Bank上进行BLAST搜索,同源性分析。并将实验序列和NCBI上下载的与该序列存在同源性的序列用软件MEGA 7.0进行比对并构建进化树。【结果】从各类样本中成功地提取到了基因组总DNA,并成功扩增出了用于动物种属鉴定的12S rRNA基因片段、Cyt b基因片段。测序分析结果表明用12S rRNA基因片段和Cyt b基因片段均可准确鉴定黑熊、藏羚羊、鬣羚、穿山甲样本的动物种属,但12S rRNA基因片段无法准确鉴定北山羊样本,BLAST搜索结果为其近缘物种山羊,而Cyt b基因片段却可准确鉴定北山羊。各样本基于12S rRNA、Cyt b基因序列构建进化树的结果与BLAST搜索结果相符。【结论】单一位点基因标记能够准确鉴别亲缘关系较远的物种。但对某些近缘物种,单一位点基因标记的鉴别能力较差,故野生动物物种鉴定宜选用2个以上的基因标记位点。

【Objective】To study the effectiveness of gene markers for wildlife species identification,we compared the differences between mt DNA 12 S rRNA and Cyt b gene markers in wildlife species identification.【Method】The isolation of DNA from wildlife samples was carried out. Partial mitochondrion DNA 12 S rRNA and Cyt b genes were amplified and sequenced. The original animal identifications of different wildlife samples were determined by BLAST search of the sequences in Gen Bank. Then,genetic distance and phylogenetic tree were analyzed by MEGA 7.0.【Result】The genome DNAs were successfully extracted from wildlife samples and partial mitochondrion DNA 12 S rRNA and Cyt b genes were amplified. For the identification of Ursus thibetanus,Pantholops hodgsonii,Capricornis milneedwardsii,and Manis javanica sample species,both the 12 S rRNA gene marker and Cyt b gene marker can identify the wild species successfully. However,in terms of identification of Capra ibex,Cyt b gene marker could distinguish Capra ibex from Capra hircus gene sequences,while 12 S rRNA gene marker not. Phylogenetic tree constructed based on 12 S rRNA and Cyt b sequences was consistent with BLAST analysis. 【Conclusion】Most of wild animals can be identified by single locus marker. When comes to some closely relative species,the identification ability of single locus marker is poor,thus two or more gene marker loci are recommended to identify wild animal species.