基于ITS-1序列的5种兔球虫系统进化分析

利用核糖体DNA内转录间隔区1(ITS-1)序列对兔艾美耳球虫进行系统进化分析,并探讨生物学和形态学特征在兔球虫进化中的意义。单卵囊分离大型、黄、肠、中型及穿孔艾美耳球虫卵囊,接种无球虫兔获得纯种卵囊,CTAB法提取卵囊基因组DNA,PCR扩增ITS-1区后克隆、测序。将测序结果与GenBank发布的兔球虫ITS-1序列进行比对和遗传距离分析,绘制系统进化树。结果显示:大型、黄、肠、中型及穿孔艾美耳球虫河北株ITS-1序列分别长320、330、351、336和341bp。5种兔球虫河北株与GenBank中同种兔球虫ITS-1序列相似性分别为96.9%、97.3%、96.9%、99.1%和99.4%。兔球虫形成单系群,该单系群分为2个姐妹谱,与卵囊残体有无相对应,其它形态学和生物学特征与系统进化无相关性。研究结果表明外残体的有无可作为兔球虫进化分类的特征。

Phylogenetic Analysis of Five Rabbit Eimeria Species Based on the ITS-1 Sequence

This experiment was conducted to study the correlation of morphological and biological features to the phylogeny of rabbit coccidia.Five species of rabbit coccidia were isolated by single-oocyst separation technology and proliferated.Genomic DNA was extracted from their sporulated oocysts by the method of CATB.The ITS-1 regions were cloned and sequenced,then they were aligned with corresponding sequence in the GenBank.The phylogenetic tree was obtained by NJ method.The complete ITS-1 sequences of E.magna HB,E.flavescens HB,E.intestinalis HB,E.media HB,E.perforans HB were 320,330,351,336 and 341 bp respectively in length.The sequence identities displayed 96.9%,97.3%,96.9%,99.1% and 99.4%.Based on the ITS-1sequence data,the rabbit coccidia monophyly was divided into 2 sister lineages,which was corresponding to the presence/absence of the oocyst residuum.Other morphological or biological traits(oocyst shape and size,pathogenicity,infection site,pre-patent and patent periods,and number of asexual generations) do not explicitly correlate with the phylogeny of rabbit coccidia.The presence/absence of oocyst residuum is an evolutionarily conserved feature.