目的 分析印度华首线虫的核糖体基因和线粒体基因序列,探讨其作为分子标记的应用潜力。方法 2016年6月,于湖南省长沙市长沙县榔梨镇捕捉到2条野生大王蛇,并从蛇体内分离获得7条印度华首线虫。提取7条印度华首线虫基因组DNA,PCR扩增核糖体DNA内转录间隔区1和2（ITS-1和ITS-2）、5.8S、线粒体DNA细胞色素c氧化酶亚基1（cox1）、核糖体RNA小亚基12S、ATP酶亚基6（atp6）、烟酰胺腺嘌呤二核苷酸脱氢酶亚基4（nad4）基因,测序。采用ClustalX 1.83软件拼接各基因测序结果,并进行多序列比对。基于cox1、12S、atp6、nad4线粒体基因序列,以旋盘尾丝虫为外群,采用贝叶斯（BI）法构建系统进化树,分析7条印度华首线虫PCR扩增序列的种内变异。结果 ITS-1、5.8S和ITS-2扩增产物长度约为900 bp,cox1、12S、atp6和atp4扩增产物长度依次约为400、600、500和400 bp。ITS-1、5.8S和ITS-2的A + T含量为52.6%~53.7%,高于G + C含量（46.3%~47.4%）,cox1、12S、atp6和atp4的A + T含量分别为67.1%~68.9%、76.9%~77.4%、73.0%~75.5%和70.3%~73.3%,均高于其G + C含量（31.1%~32.9%、22.6%~23.1%、24.5%~27.0%和26.7%~29.7%）。ITS-1、5.8S、ITS-2、cox1、12S、atp6和atp4的种内变异依次为0~2.5%、0、0~5.0%、0~2.6%、0~3.7%、0.7%~8.7%和0~11.0%。7条印度华首线虫线粒体基因序列聚于一支,与钩口科锡兰钩口线虫的亲缘关系最近。结论 印度华首线虫核糖体基因序列中ITS-2变异程度最大,有望成为研究印度华首线虫种内变异的理想分子标记。线粒体基因序列变异程度从低到高依次为cox1 < 12S < atp6 < nad4,nad4是研究印度华首线虫遗传多样性的理想线粒体分子标记。

Objective To analyze the ribosomal and mitochondrial genes sequences polymorphisms of Kalicephalus indicus, and discuss their application potential as molecular markers. Methods Two wild Elaphe carinata snakes were captured in June 2016, in Zhangli Town, Changsha County, Hunan Province, and seven K. indicus adult worms were isolated from the snakes. Total DNA from the worms was extracted. The sequences of the first and second internal transcribed spacer regions 1 and 2(ITS-1 and ITS-2), 5.8S, mitochondrial cytochrome c oxidase subunit 1 (cox1), small subunit 12S of ribosomal RNA, ATP synthase subunit 6 (atp6), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (nad4) of K. indicus were amplified using PCR and subjected to DNA sequencing. The sequencing results of each gene were aligned using ClustalX 1.83. A phylogenetic tree based on the mitochondrial DNA sequences cox1, 12S, atp6 and nad4 was constructed using Bayesian method, using Onchocerca volvulus as an outgroup. Intraspecific variation in these sequences among the seven K. indicus samples were analyzed. Results The size of the amplified products of ITS-1, 5.8S and ITS-2 was approximately 900 bp. The size of the amplified products of cox1, 12S, atp6 and nad4 was approximately 400, 600, 500 and 400 bp, respectively. The content of A + T of ITS-1, 5.8S and ITS-2 is 52.7%-53.7%, higher than its content of G + C (46.3%-47.4%). The content of A + T of cox1, 12S, atp6 and nad4 is 67.1%-68.9%, 76.9%-77.4%, 73.0%-75.5%, and 70.3%-73.3%, respectively, higher than their content of G + C (31.1%-32.9%, 22.6%-23.1%, 24.5%-27.0% and 26.7%-29.7%). Intraspecific variation in ITS-1, 5.8S, ITS-2, cox1, 12S, atp6 and nad4 were 0-2.5%, 0, 0-5.0%, 0-2.6%, 0-3.8%, 0.7%-8.7% and 0-11.0%, respectively. The phylogenetic tree clustered the seven K. indicus individuals into the same branch, with Ancylostoma ceylanicum being the closest species. Conclusion The ITS-2 sequence with the largest variation is expected to be an ideal molecular marker for studying the intraspecific variation of K. indicus. The degree of its intraspecific variation in mitochondrial gene sequences is as follows: cox1 < 12S < atp6 < nad4, and nad4 is an ideal mitochondrial molecular marker for studying the genetic diversity of K. indicus.