弓首蛔虫及狮弓蛔虫线粒体基因组nad4基因多态性研究

目的比较犬、猫常见蛔虫的线粒体DNA(mtDNA)中烟酰胺脱氢酶亚基Ⅳ基因(nad4)部分序列(pnad4),以期找出它们之间的序列差异和种群遗传关系。方法抽提61个弓首属蛔虫(包括犬弓首蛔虫、猫弓首蛔虫、马来西亚弓首蛔虫和犊弓首蛔虫)和狮弓蛔虫虫体的总DNA,用PCR方法扩增mtDNApnad4,然后用SSCP技术和DNA测序对序列进行分析研究。结果犬弓首蛔虫种群内pnad4的序列差异为1.17%,与猫弓首蛔虫、马来西亚弓首蛔虫、牛弓首蛔虫和狮弓蛔虫种间的平均序列差异分别为13.92%、16.48%、15.12%和20.32%。猫弓首蛔虫种群内pnad4的序列差异为1.00%,与马来西亚弓首蛔虫、牛弓首蛔虫、狮弓蛔虫平均序列差异分别是17.90%、13.55%和18.50%。马来西亚弓首蛔虫种群内pnad4序列差异为0.13%,与牛弓首蛔虫、狮弓蛔虫序列差异分别为13.50%和20.60%。结论犬弓首蛔虫不同地方虫株的pnad4有一定差异,猫弓首蛔虫种群内差异较小,马来西亚弓首蛔虫种群内差异很小。弓首属各虫种之间的差异以及与狮弓蛔虫的差异都较大。马来西亚弓首蛔虫pnad4序列与其它虫种差异都较大,证明它确为一独立种。pnad4序列是弓首蛔虫和狮弓蛔虫理想的种特异的遗传标记。

Analysis of DNA Polymorphism in Mitochondrial NADH Dehydrogenase Subunit 4 in Toxocara Species and Toxascaris leonina

Objective To analyze the genetic relationship and the variation of the mitochondrial NADH dehydrogenase subunit 4 (nad4) genes in Toxocara species and Toxascaris leonina. Methods Genomic DNA from 61 samples of ascaridoid nematodes (Toxocara canis, Toxocara cati, Toxocara malaysiensis, Toxocara vitulorum and Toxascaris leonina) were collected from different geographical locations. A portion of the mitochondrial NADH dehydrogenase subunit 4 gene (pnad4) from each sample was amplified by polymerase chain reaction (PCR). Sequence heterogeneity in the pnad4 was examined and compared using single-strand conformation polymorphism (SSCP) analysis, followed by DNA sequencing analysis. Results The variation of pnad4 gene within the species of T. canis,T. cati, and T. malaysiensis was 1.17%, 1.00% and 0.13%, respectively. In the inter-species comparison, the differences of pnad4 gene were 13.92% between T.canis and T.cati,16.48% between T.canis and T. malaysiensis, 15.12% between T. canis and T. vitulorum, 20.32% between T. canis and T. leonina, 17.90% between T. cati and T. malaysiensis, 13.55% between T. cati and T. vitulorum, 18.50% between T. cati and T. leonina, 13.50% between T. malaysiensis and T. vitulorum, and 20.60% between T. malaysiensis and T. leonina. Conclusion The intra-species variation of pnad4 was lower than the inter-species variation. The results also suggested that T.malaysiensis is a distinct group of nematode, and pnad4 may be used as a genetic marker for the species identification. The results may also be used for the study of molecular systematics, population genetic structures and the molecular ecology of Toxocara and Toxascaris species.