鸡蛔虫凉山州分离株ITS和5.8SrDNA序列测定及种系发育分析

应用保守引物BD1和BD2对7个鸡蛔虫凉山州分离株的核糖体DNA内转录间隔区(ITS)及5.8S rDNA序列进行PCR扩增和序列测定,并用ITS-1、ITS-2序列重构鸡蛔虫与其他蛔虫的系统发育关系。测序结果显示所获得的鸡蛔虫ITS及5.8S rDNA序列大小为974~989 bp,同源性为98.9%~100.0%。其中ITS-1、5.8S rDNA和ITS-2片段大小分别为473~481、157和337~359 bp,同源性分别为98.5%~100.0%、100.0% 和98.5%~100.0%。系统发育树显示所有鸡蛔虫分离株聚在同一分支,能与其他蛔虫相区别。研究结果表明,鸡蛔虫的ITS-1、ITS-2序列种内变异小,但种间差异大,故可作为分子标记用于鸡蛔虫的虫种鉴定,为鸡蛔虫的分子分类、分子流行病学调查和种群遗传的进一步研究奠定基础。

Sequencing and Phylogenetic Analysis of ITS and 5.8S rDNA of Ascaridia galli Isolated from Liangshan

The internal transcribed spacer (ITS) and 5.8S ribosomal DNA (5.8S rDNA) of roudworm (Ascaridia galli, A.galli) samples collected from Liangshan,Sichuan were amplified by PCR using a pair of conserved primers (BD1 and BD2),then sequenced and analyzed the sequences,phylogenetic trees based on ITS-1 and ITS-2 were reconstructed using Mega 5.0 software.The results showed that ITS and 5.8S rDNA sequences of Ascaridia galli were 974 to 989 bp,with homology among these sequences was 98.9% to 100.0%.The sequences of ITS-1,5.8S rDNA and ITS-2 were 473 to 481,157 and 337 to 359 bp,respectively.With homologies of 98.5% to 100.0%,100.0% and 98.5% to 100.0%,respectively.The phylogenetic trees showed that all the Ascaridia galli isolates were formed the same cluster,and could identify with other Ascaridida.The results indicated that there was no significant variation in ITS-1 and ITS-2 sequences within Ascaridia galli,while inter-species difference was obvious,so they could be used as genetic marker to identify the species of Ascaridia galli,and provided the foundation for further research on molecular classification,epidemiological survey and population genetics of Ascaridia galli.