黑熊源蛔虫的分子鉴定及其线粒体pcox1、pnad5基因的系统发育分析
Molecular identification and phylogenetic analysis of sequences of mitochondrial pcox1 and pnad5 gene of ascarid isolates from black bear
陈淞楠 1邱启官 2阳朝伟 2何善逵 2李志康 1李宁倩 1刘伟1
作者信息
- 1. 湖南农业大学 动物医学院,湖南 长沙 410128
- 2. 长沙生态动物园,湖南 长沙 410029
- 折叠
摘要
为鉴定黑熊源蛔虫的种类及分析其系统的发育特征,本实验对长沙生态动物园黑熊肠道内分离的6株蛔虫采用PCR扩增其线粒体部分cox1基因(pcox1)和部分nad5基因(pnad5)并测序,采用DNAStar 5.0软件分析这两种基因与GenBank中不同蛔虫相应基因序列的同源性;利用DnaSP 5.0软件分析pcox1和pnad5基因的单倍型数量、核苷酸多样性以及序列之间的核苷酸差异;并采用MEGA7软件构建二者的进化树分析黑熊源蛔虫的遗传进化关系.PCR结果显示,6株黑熊源蛔虫pcox1、pnad5基因分别为408 bp和528 bp,两种基因序列之间的同源性分别为99.30%～100%和99.20～100%,与GenBank中转移贝蛔虫相应基因序列的同源性分别为96.80%～99.80%和95.10%～95.30%.DnaSP 5.0软件分析结果显示,黑熊源蛔虫pcox1基因共检测到5个单倍型,单倍型多样性和核苷酸多样性分别为0.933±0.015和0.00675,核苷酸差异平均值为2.733;pnad5基因共检测到6个单倍型,单倍型多样性和核苷酸多样性分别为1.000±0.00926和0.00756,平均核苷酸差异为4.000.基于pcox1和pnad5基因构建的系统进化树显示,6株黑熊源蛔虫均与转移贝蛔虫聚为同一分支;与贝氏西蛔虫聚为一大支,与其他蛔虫遗传距离较远.上述结果表明6株黑熊源蛔虫均为转移贝蛔虫,且他们的遗传变异程度均较低,pcox1基因和pnad5基因均可作为转移贝蛔虫分类鉴定的分子标记.本研究首次对黑熊源蛔虫进行分子鉴定并分析其的遗传进化关系,为转移贝蛔虫的分类鉴定以及野生黑熊蛔虫病的防治提供参考.
Abstract
In order to identify the species of ascarid isolated from black bear and analyze their phylogenetic relationship,this experiment used PCR to amplify part of mitochondrial cox1 gene(pcox1)and part of nad5 gene(pnad5)from six strains of roundworms isolated from the intestine of black bear in Changsha Eco-zoological Zoo.The PCR products were then subjected to sequencing.DNAStar 5.0 software was used to analyze the homology between these two genes and the corresponding gene sequences of different ascarid in GenBank,and MEGA7 software was used to construct an evolutionary tree of the two genes to analyze the genetic evolutionary relationship of black bear-derived ascarid.The results showed that the lengths of pcox1 and pnad5 were 408bp and 528bp,respectively,and the homologies among the 6 strains were 99.3-100.0%and 99.0-100.0%,respectively.Compared with pcox1 and pnad5 sequences of other Baylisascaris transfuga in GenBank,the homologies were 96.8-99.8%and 95.1-95.3%,respectively.DnaSP 5.0 software analysis showed that a total of five haplotypes were detected in the pcox1 gene of six isolates from black bear,with haplotype diversity and nucleotide diversity of 0.933±0.015 and 0.00675,respectively,and a mean nucleotide difference of 2.733;six haplotypes were detected in the pnad5 gene,with haplotype diversity and nucleotide diversity of 1.000±0.00926 and 0.00756,respectively.0.00926 and 0.00756,with a mean nucleotide difference of 4.000.The phylogenetic tree constructed based on the pcox1 and pnad5 genes showed that the six isolates were clustered in the same branch as Baylisascaris transfuga isolates.This branch was clustered with Baylisascaris schroederi clade,and was far from the other ascarid.The results indicated that all six isolates from black bear(Ursus thibetanus)were Baylisascaris transfuga and they all have a low level of genetic variation,both pcox1 and pnad5 genes could be used as molecular markers for the taxonomic identification of Baylisascaris transfuga.This study is the first to identify and analyse the genetic-evolutionary relationships of bear-derived ascarid.This study provides a theoretical basis for the taxonomic identification of Baylisascaris transfuga,and the prevention and control of ascariasis in wild black bears.
关键词
黑熊源蛔虫/cox1/nad5/分子鉴定/系统发育分析Key words
black bear-derived ascarid/cox1/nad5/molecular identification/phylogenetic analysis引用本文复制引用
基金项目
湖南省自然科学基金(2021JJ30335)
出版年
2024
NETL
NSTL
万方数据