细口杯环线虫形态学和分子特征分析

doi:10.16431/j.cnki.1671-7236.2019.01.032

摘要/Abstract

摘要：

为探讨细口杯环线虫（Cylicocyclus leptostomum）的分类地位和系统发育关系，本试验利用数码显微镜对细口杯环线虫进行了形态观察，运用PCR扩增其核糖体DNA内部转录间隔区（rDNA-ITS）序列，并从GenBank中下载12种圆线虫的ITS序列，以马圆形线虫（Strongylus equinus）为外群，运用最大似然法（maximum likelihood，ML）构建系统发育树。结果显示，细口杯环线虫中等大小，口囊呈圆柱形，宽度大于深度，口囊底部有小齿；口囊壁前端薄，后端基部有明显的环箍形增厚；外叶冠由20~24个小叶组成，内叶冠由50~60个小叶组成；食道漏斗较小；雄虫生殖锥较长，呈圆锥形；雌虫尾部直，尾尖呈指形；所测ITS序列总长度为837 bp，其中ITS1长366 bp，5.8S长153 bp，ITS2长318 bp；ITS1的GC含量（46.0%）明显高于ITS2（39.8%）；经BLAST同源性比对分析，本研究线虫ITS序列与GenBank上登录的同种线虫序列（登录号：AJ004849、Y08587）同源性达99.85%，与阿氏杯环线虫的同源性达99.0%，与杯环属内其他线虫的同源性仅为93.33%~98.45%；ITS序列种间差异远大于种内差异；系统进化分析显示，细口杯环线虫与阿氏杯环线虫的亲缘关系较近，而与杯环属内其他线虫的亲缘关系相对较远。综上所述，ITS序列可作为鉴定寄生线虫的分子遗传标记，证实所采标本是细口杯环线虫，并首次在国内报道了细口杯环线虫的ITS序列，为该线虫的进一步研究奠定基础。

关键词: 细口杯环线虫; 形态描述; 核糖体DNA内部转录间隔区(rDNA-ITS); 系统发育分析

Abstract:

In order to investigate the taxonomic status and phylogenetic relationship of Cylicocyclus leptostomum (C.leptostomum),this study used a digital microscope to observe the morphology of C. leptostomum,the ribosomal DNA internal transcribed spacer (rDNA-ITS) sequence was amplified by PCR,and 12 other ITS sequences of Strongylid nematodes were downloaded from GenBank,and the phylogenetic tree was constructed by maximum likelihood (ML) using Strongylus equinus as the outer group.The results showed that the nematode was medium-sized,the cyst was cylindrical,the width was greater than the depth,with small teeth at the bottom;The front end of the pouch wall was thin,and the base of the back end had obvious hoop-shaped thickening;The outer leaf crown was composed of 20 to 24 leaflets,and the inner leaf crown was composed of 50 to 60 leaflets;The esophagus funnel was smaller;Male reproductive cone was longer,conical;The female tail was straight,tail tip was finger-shaped;The total length of the measured ITS sequence was 837 bp,of which ITS1 was 366 bp,5.8S was 153 bp,and ITS2 was 318 bp.The GC content of ITS1 (46.0%) was significantly higher than that of ITS2 (39.8%);By BLAST homology alignment analysis,the ITS sequence of this study was 99.85% homologous with the same nematode sequence registered in GenBank (accession No.:AJ004849 and Y08587),and the homology with C.ashworthi,up to 99.0%,the homology with other nematodes in the genus was only 93.33% to 98.45%.The interspecies differences in ITS sequences were much larger than intraspecies differences.Phylogenetic analysis showed that C.leptostomum was closely related to C.ashworthi,and it was relatively distant from other nematodes in the genus.In summary,the ITS sequence could be used as a molecular genetic marker for identifying parasitic nematodes.It was confirmed that the specimen was a C.leptostomum,and the ITS sequence of C.leptostomum was reported for the first time in China,which would lay a foundation for further studies on this species.

Key words: Cylicocyclus leptostomum; morphological description; rDNA-ITS; phylogenetic analysis

中图分类号:

S852.73⁺1

胡丽莉, 孙彦, 刘莹莹, 王晨, 卜艳珍. 细口杯环线虫形态学和分子特征分析[J]. 《中国畜牧兽医》, 2019, 46(1): 271-278.

HU Lili, SUN Yan, LIU Yingying, WANG Chen, BU Yanzhen. Morphological and Molecular Characteristics Analysis of Cylicocyclus leptostomum[J]. , 2019, 46(1): 271-278.

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