中国寄生虫学与寄生虫病杂志

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陶氏颚口线虫成虫的扫描电镜观察及基于ITS2和COX1基因的系统发生分析

李雯雯1,2,任怡静2,李健3,黄维义2,4 *   

  1. 1 中山大学附属第一医院动物实验中心,广州 510080;2 广西大学动物科学技术学院,南宁 530005;3 复旦大学生命科学学院微生物学与微生物工程系,上海 200433;4 广西大学食品质量与安全研究中心,南宁 530005
  • 出版日期:2015-04-30 发布日期:2015-05-04

Scanning Electron Microscopic Observation on Adult Gnathostoma doloresi Worms and the Phylogenetic Analysis of G. doloresi Based on ITS2 and COX1 Gene Sequences

LI Wen-wen1,2,Ren Yi-jing2,LI Jian3,HUANG Wei-yi2,4 *   

  1. 1 Laboratory Animal Center,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou 510080,China;2 College of Animal Science and Technology,Guangxi University,Nanning 530005,China;3 Department of Microbiology and Microbial Engineering,School of Life Sciences,Fudan University,Shanghai 200433,China;4 Research Center for Food Safety and Quality,Guangxi University,Nanning 530005,China
  • Online:2015-04-30 Published:2015-05-04

摘要:

目的  观察野猪体内分离的陶氏颚口线虫(Gnathostoma doloresi)成虫体表的超微结构,并基于其核糖体第二内转录间隔区(ITS2)和细胞色素氧化酶亚基I(COX1)基因分析其分子系统发生关系。  方法  用戊二醛和锇酸双固定法固定从野猪胃壁分离的陶氏颚口线虫成虫2条,扫描电镜下观察其体表超微结构。PCR法分别扩增成虫ITS2和COX1基因并测序,测序获得的序列经校正处理后使用BLAST在线工具进行比对,并运用MEGA 6.0软件将所测序列与GenBank中颚口线虫属相关序列进行多重比对,采用邻位相连法构建系统发生树。  结果  扫描电镜结果显示,陶氏颚口线虫成虫呈酒瓶状,全身布满小棘;头球呈扁球体状,头部与体部间凹陷成一颈沟,沟内无棘;颈乳突和体中部膨大区域的体棘形状区分明显。所获ITS2和COX1基因序列的长度分别为418和381 bp,与GenBank中陶氏颚口线虫的ITS2(登录号AB181156)和COX1(登录号AB180100)基因的序列一致性分别为99%和98%,提交至GenBank获得登录号分别为JN408329和JN408299。ITS2和COX1种系发生树结果显示,2条陶氏颚口线虫独立分支自展值分别为100%和85%,与刚刺颚口线虫(G. hispidum)也分别构成自展值为99%和93%的独立分支。  结论  本研究为陶氏颚口线虫成虫的扫描电镜形态和系统发生关系提供了资料。

关键词: 陶氏颚口线虫, 扫描电镜, 核糖体第二内转录间隔区, 细胞色素氧化酶亚基Ⅰ, 种系发生分析

Abstract:

Objective  To observe the ultrastructure of adult Gnathostoma doloresi worms isolated from wild boar by using scanning electron microscope(SEM), and analyze its phylogenetic relationships based on ITS2 and COX1 gene sequences.  Methods  Two adult G. doloresi worms were fixed by glutaraldehyde and osmium peroxide. Ultrastructural characters of those samples were observed under SEM. Amplification and sequencing of the ITS2 and COX1 genes were performed following the extraction of total genomic DNA. Sequence analysis was performed based on multiple alignments and phylogenetic analysis was made by Neighbor-Joining method using MEGA 6.0.  Results  The bottle-shaped adult worm covered with numerous small spines. The cervical groove connected head bulb and body without spines. There was obvious distinction in body spines which surround cervical papillae and swollen area in the middle part of the body. The fragments of ITS2(418 bp) and COX1(381 bp) gene were obtained by PCR combined with sequencing, and were registered to the GenBank database with the accession No. of JN408329 and JN408299, respectively. The BLAST results showed that, two sequences had 99% and 98% consistency with G. doloresi ITS2 (GenBank accession No. AB181156) and COX1 (No. AB180100) gene sequences, respectively. The phylogenetic tree indicated that the two G. doloresi worms were at the same clade with a bootstrap value at 100% and 85% based on the ITS2 and COX1 sequences, respectively. G. doloresi and G. hispidum were also clustered together.  Conclusion  The results provide adequate information for the SEM morphological data of adult G. doloresi worms, and its phylogenetic relationship.