3种兔球虫ITS-1序列测定与系统发育分析

Sequence and phylogenetic analysis of ITS-1 rDNA from three species of rabbit coccidia

从河北省兔场分别单卵囊分离孢子化大型艾美耳球虫卵囊、黄艾美耳球虫卵囊及肠艾美耳球虫卵囊,接种无球虫兔后获得纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18SrDNA和5.8SrDNA保守引物,PCR扩增3种兔球虫ITS-1片段,产物纯化后测序。将3种球虫ITS-1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比较,绘制系统发育树。结果表明,大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫河北株分别扩增出424、455、434bp的ITS-1片段。大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫河北株与GenBank中发布的同种兔球虫ITS-1序列相似性分别为97.4%、97.9%和96.9%。系统发育树显示兔球虫ITS-1序列形成1个单系群,该单系群根据寄生部位分为2个姊妹群。

To further determine E. magna, E. flavescens and E. intestinalis at the molecular level, three species of rabbit coccidia were isolated by single-oocyst separation technology from rabbitries in Hebei and were proliferated. Genomic DNA were extracted from their sporulated oocysts by the method of CATB. Using conservative primer of 18S rDNA and 5.8S rDNA of Eirneria, ITS-1 rDNA gene fragments were amplified and sequenced, then they were analysed by DNASTAR and aligned with corresponding sequence of theeleven species of rabbit-infecting Eimeria in the GenBank. The phylogenetic tree was obtained using MEGA4.0. The results indicated that the gene fragments of E. magna, E. flavescens and E. intestinalis were respectively amplified with 424,455 and 434 bp. Sequence alignment showed that percentage similarity displayed 97.4 %（ E. magna in Geneback vs E. magna HB ）, 97.9 % （ E. flavescens in Geneback vs E. flavescens. HB）, 96.9 % （E. intestinalis in Geneback vs E. intestinalis HB）, three species of rabbit coccidia from Hebei with eleven species of rabbit-infecting Eirneria in the GenBank were located in a monophyletic, which was divided into 2 sister lineages,corresponding to the parasitic location.