基于DNA条形码技术对青海鼠疫疫源地内主要媒介蚤的分子鉴定研究

Molecular identification of main vector fleas in Qinghai plague foci by DNA barcoding

目的应用DNA条形码技术对青海省鼠疫疫源地内主要媒介蚤进行分子鉴定，弥补蚤类传统形态分类方法的不足。方法于青海省3个州、2个市、5个县采集36份蚤种肌肉组织，PCR方法扩增细胞色素c氧化酶亚基I（coI）基因片段并进行测序和比对；用Mega6软件（K2-P双参数模型）计算种内及种间遗传距离，以邻接法（neighbor-joining，NJ）构建系统发育树。结果共测得2总科4属6种媒介蚤36条COI基因部分序列。全部蚤种平均遗传距离为0．119。细钩盖蚤种内遗传距离最小（0．002），斧形盖蚤最大（0．027）。斧形盖蚤和细钩盖蚤的种间遗传距离最小（0．039），人蚤和细钩盖蚤距离最大（0．207）。种间遗传距离显著大于种内遗传距离。NJ树显示，同一物种的不同个体均形成高支持率的单系，种间分支明显。结论DNA条形码技术适用于青海省鼠疫疫源地内主要媒介蚤种的分子鉴定，可弥补蚤类传统形态分类方法的不足。

Objective To make up the limitations of traditional morphological classification methods, we identified vector fleas by DNA barcoding in Qinghai Province. Methods The mt DNA cytochrome c oxidase subunit I （CO I ） gene was amplified by PCR from 36 muscle tissues of fleas in 3 states, 2 cities and 5 counties of Qinghai Province, and the obtained CO I gene fragments were sequenced and aligned. The intra- and inter-species genetic distances were calculated with Mega 6 software using K2-P model and a phylogenetic tree was constructed with neighbor-joining （NJ） method. Results Totally 36 CO I gene sequences of 2 superfamilies, 4 genera and 6 kinds of vector fleas were measured, the average genetic distance was 0.119, and the intraspecific distance was 0.002 - 0.027, the interspecific distance was 0.039 - 0.207, and the interspecific genetic distance was significantly greater than the intraspecific genetic distance. NJ tree showed the same species had formed a single line with high support rate and interspecific branch was clear. Conclusion DNA barcoding is suitable for identification of vector fleas in Qinghai Province, may make up the limitations of traditional morphological classification methods.