褐黄血蜱与铃头血蜱的分子生物学鉴定

Molecular identification of Haemaphysalis flava and Haemaphysalis campanulata

目的建立2种形态学特征非常相近的褐黄血蜱及铃头血蜱的分子生物学鉴定方法,探讨它们的系统发生关系。方法在湖北省从动物体表采集寄生蜱,形态学鉴定完毕后,采用PCR方法从2种血蜱基因组中扩增12SrDNA、16SrDNA及线粒体细胞色素氧化酶亚基Ⅰ(COⅠ)基因,测序后进行同源性分析,用PAUP4.0软件分别构建系统发生树并进行系统进化分析。结果 2种血蜱12SrDNA、16SrDNA及COⅠ基因之间的同源性分别为90.8%、90.4%和86.8%,湖北省采集的褐黄血蜱3个基因片段与已知褐黄血蜱的同源性分别为100%、99.5%和99.7%。用这2种蜱的12SrDNA、16SrDNA及COⅠ基因的核苷酸序列分别构建系统发生树,湖北省采集的褐黄血蜱与已知的褐黄血蜱聚在一起,褐黄血蜱及铃头血蜱均形成独立的分支,但用3个基因构建的系统发生树中血蜱属不同种间的亲缘关系不同。结论对于形态学特征相近蜱种的鉴定,在传统形态学分类的基础上结合分子生物学鉴定方法能更准确地鉴定蜱的种类,也能更好地了解其进化关系,为蜱传播疾病的预防控制提供依据。

Objective To establish a method for identifying Haemaphysalis flava and H. campanulata with molecular markers, and to learn the phylogenetic relationship of these two species of ticks. Methods Ticks were collected from animals in Hubei province, and identified by their morphological characteristics. The 12S rDNA, 16S rDNA and mitochondrial cytochrome oxidase subunitⅠ gene （COⅠ） from the genomes of the two kinds of ticks were amplified by PCR and sequenced for homology analysis. PAUP 4.0 software was used to construct the individual phylogenetic trees of the three gene sequences for phylogenetic analysis. Results The identities between the two species of ticks were 90.8% for 12S rDNA, 90.4% for 16S rDNA, and 86.8% for COⅠ, with the homology of the three gene fragments between collected H. flava and known H. flava being 100%, 99.5% and 99.7%, respectively. On the phylogenetic trees based on the nucleotide sequences of the 12S rDNA, 16S rDNA and COⅠ gene, H. flava was clustered together with their respective class. H. flava and the H. campanulata formed a distinct branch. However, the phylogenetic relationships between different species of Haemaphysalis were different on the trees based on these three genes. Conclusion As far as identification of the ticks with similar morphological features is concerned, molecular markers in combination with the conventional morphological classification can make it more accurate, contributing to better understanding of their evolutionary relationships. Further phylogenetic analysis of gene sequences also provides a basis for the prevention and control of tick-borne diseases.