湖南省不同宿主犬弓首蛔虫线粒体pcox1和pnad1基因序列遗传变异的研究

Genetic analysis of mitochondrial pcox1 and pnad1 genes of Toxocara canis isolated from different hosts in Hunan province

为探究湖南省不同宿主来源犬弓首蛔虫的种内遗传变异情况,本研究利用PCR法对14株分离自湖南省部分地区不同宿主(犬和狼)源犬弓首蛔虫分离株线粒体细胞色素c氧化酶第I亚基(cox1)基因部分序列(pcox1)和烟酰胺腺嘌呤二核苷酸脱氢酶亚单位I(nad1)基因部分序列(pnad1)进行PCR扩增与测序,分析该两个序列的同源性,并用pcox1和pnad1基因序列分别构建不同宿主源犬弓首蛔虫与其他蛔虫的种系发育树。结果显示,本研究获得的犬和狼源弓首蛔虫分离株pcox1和pnad1基因序列大小分别为442 bp和527 bp,其种内遗传变异率分别为0~1.7%和0.2%~2.2%,与已知不同宿主源(犬、人、狐狸等)弓首蛔虫分离株对应核苷酸序列同源性分别为94.6%~99.5%(pcox1)和98.4%~99.2%(pnad1);检测到二者的单倍型分别为13个和12个,单倍型多样性分别为0.989和0.967,核苷酸多样性分别为0.01675和0.00509。基于两种基因构建系统发育树的分析结果一致,本研究获得的14个不同宿主来源犬弓首蛔虫分离株与GenBank已有犬弓首蛔虫分离株位于同一分支,与猫弓首蛔虫和马来西亚弓首蛔虫分离株距离较近,与其他蛔虫所属分支距离较远。本研究结果表明,不同宿主源犬弓首蛔虫湖南分离株遗传变异程度较低,且pcox1基因较pnad1基因序列更适合作为犬弓首蛔虫分离株遗传变异研究的分子标记。本研究为不同宿主源弓首蛔虫的分子流行病学调查及种群遗传学研究奠定了基础。

To investigate the genetic variations of Toxocara canis isolates from different hosts in Hunan province,China,the partial sequences of mitochondrial cytochrome I(cox1)gene and nicotinamide adenine dinucleotide dehydrogenase subunit I(nad1)gene of T.canis isolated from dogs and wolves were amplified using polymerase chain reaction method and sequenced to analyze the genetic homology,respectively.Phylogenetic trees of the sequenced genes and their corresponding reference strains were constructed.The results showed that the length of pcox1 and pnad1 gene sequences of T.canis isolated from different hosts were 442 bp and 527 bp,respectively.The intraspecific genetic differences were 0-1.7%and 0.2%-2.2%,respectively.The sequence similarity compared with T.canis isolated from different hosts(dog,human,and fox)that available in GenBank were 94.6%-99.5%(pcox1)and 98.4%-99.2%(pnad1),respectively.13 and 12 haplotypes were identified in pcox1 and pnad1 genes,respectively,with the average haplotype diversity of 0.989 and 0.967,respectively.The nucleotide diversities of pcox1 and pnad1 genes were 0.01675 and 0.00509,respectively.Phylogenetic analysis based on these two genetic markers were similar.14 specimens obtained in the present study were in the same branch as the T.canis isolates from different hosts,and these strains were showing closer genetic relationship to Toxocara cati and Toxocara malaysiensis isolates.These results showed that the isolates in this study were exhibiting low genetic variation degrees,and the mitochondrial pcox1 gene would be a better candidate as a molecular marker for the studying of genetic variation.The study provided a foundation for the molecular epidemiology and genetic investigation of T.canis from different hosts.