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为了研究黑斑蛙裂头蚴种系发育关系,本研究利用聚合酶链式反应(PCR)对黑斑蛙裂头蚴湖南株核糖体DNA内转录间隔区(ITS)进行扩增与测序,应用ClustalX 2.0程序对序列进行比对,再利用Mega4.0程序进行NJ法绘制种系发育树,研究黑斑蛙裂头蚴与其他带科绦虫的种群遗传关系。结果显示,湖南省不同地区10个黑斑蛙裂头蚴分离株ITS序列基本一致,为1 362~1 382bp,各分离株之间ITS-1和ITS-2序列的同源性均高于95.4%和94.6%;与基因库中其他带科绦虫ITS-1和ITS-2序列的同源性均低于59.5%和56.9%。通过建立NJ系统发育树,湖南省黑斑蛙裂头蚴分离株与欧猬迭宫绦虫位于同一大分支,得到了很好的鉴定。黑斑蛙ITS序列在种内相对保守,而种间差异较大,因此ITS序列可以作为分子标记研究黑斑蛙裂头蚴种系遗传变异,从而为黑斑蛙裂头蚴的分子流行病学和群体遗传研究奠定基础。
In order to study the germline development of Spargania blackfly, we used polymerase chain reaction (PCR) to amplify and sequence the ribosomal DNA internal transcribed spacer (ITS) in Hunan Strain larvae. Using the ClustalX 2.0 program The sequences were compared and then phylogenetic tree was drawn by NJ method with Mega4.0 program to study the genetic relationship between Spider larvae of Black Spot Frog and other tapeworms. The results showed that the sequences of ITS isolates from 10 black spot frogs in different areas of Hunan Province were basically the same, ranging from 1 362 to 1 382 bp. The ITS-1 and ITS-2 homologies among all isolates were higher than 95.4% 94.6%. The homologies of ITS-1 and ITS-2 sequences to other tapeworms in GenBank were lower than 59.5% and 56.9%, respectively. Through the establishment of NJ phylogenetic tree, the Hunan black spot frog spider catarrhal isolates and the European Hedgehog intestine is located in the same branch, has been well identified. The ITS sequence of Rana nigromaculata is relatively conserved within the species and the species is highly divergent. Therefore, the ITS sequence can be used as a molecular marker to study the genetic variation of the spider larvae of R. fringe and thus provide molecular epidemiological and population genetic studies Lay the foundation.