基于16S rDNA和COⅠ基因的3种血蜱分子生物学鉴定

Identification of Haemaphysalis longicornis,H.flava and H.campanulata based on molecular markers of 16S rDNA and COⅠgene

目的 利用16S rDNA基因及线粒体DNA细胞色素氧化酶亚基Ⅰ（cytochrome oxidase subunit I gene，COⅠ）基因建立3种形态学特征相近的血蜱属蜱类-长角血蜱、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 在上海市动物体表采集寄生蜱，在解剖显微镜下进行形态学鉴定后，提取蜱样本基因组DNA，采用PCR方法从蜱基因组中扩增16S rDNA及COⅠ基因，测序后进行同源性分析，用Mega 6.0软件分别构建系统发生树并进行系统进化分析。 结果 3种血蜱中长角血蜱的16S rDNA序列和COⅠ序列与GenBank中的长角血蜱的16S rDNA序列和COⅠ序列同源性分别为96.0%-97.2%，96.9%-98.8%；褐黄血蜱的16S rDNA序列和COⅠ序列与GenBank中的褐黄血蜱的16S rDNA序列和COⅠ序列同源性分别为95.9%-98.3%、87.1%-91.9%；铃头血蜱的16S rDNA序列与GenBank中来自日本的铃头血蜱（AB819170）的16S rDNA序列同源性为97.5%；3种血蜱16S rDNA基因之间的同源性分别为88.0%、87.0%、86.9%，COⅠ基因之间的同源性分别为80.4%、80.8%、82.8%。用16S rDNA及COⅠ基因构建系统发生树中，长角血蜱、褐黄血蜱分别与已知的长角血蜱和褐黄血蜱聚在一起，长角血蜱、褐黄血蜱均形成独立的分支。 结论 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，并能更好地了解其系统发育进化关系。

Objective To establish a molecular biological method for identifying Haemaphysalis longi- cornis, H. flava and H. campanulata with markers of 16S rDNA gene and cytochrome oxidase subunit I gene （CO I ）, and analyze the phylogenetic relationship of these three species of ticks. Methods Ticks were collected from the body of animals in Shanghai, and identified by their morphological characteristics under the dissecting microscope. Then the genomic DNA was extracted from these samples and the 16S rDNA and CO I gene were amplified by PCR from the genomes of the three species of ticks and they were sequenced for ho- mology analysis. Mega 6.0 software was used to construct the phylogenetic trees of the gene sequences for phylogenetic analysis. Results In this three species of Haemaphysalis ticks, the identities of the 16S rDNA gene and CO I gene of tt. longicornis to the 16S rDNA gene and CO I gene of other isolates in GenBank were 96.0%-97.2% and 96.9%-98.8%, respectively. The identities of the 16S rDNA gene and CO I gene of H.fla- va to the 16S rDNA gene and CO I gene of other isolates in GenBank were 95.9%-98.3% and 87.1%-91.9%, respectively. The identities of the 16S rDNA gene of tt. campanulata to that from Japan （AB819170） in Gen- Bank was 97.5%. The identities between the three species of Haemaphysalis ticks were 88.0%, 87.0% , 86.9% for 16S rDNA and 80.4% ,80.8%, 82.8% for CO I . The phylogenetic trees were constructed with the nu- cleotide sequence of 16S rDNA and CO I gene among three species of ticks and other Haemaphysalis genus ticks, respectively. The results showed that H. longicornis and H.flava were clustered together respectively and they all had formed independent branches. Conclusions It can more accurately identify ticks species by combination of molecular biological identification methods and traditional morphological classification.