3种兔球虫18S rDNA部分序列测定与系统发育分析

The sequence and phylogenetic analysis of 18S rDNA from three species of rabbit coccidia

采用单卵囊分离法从河北某兔场分离大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫,接种无球虫兔后获得大量纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18S rDNA保守引物,PCR扩增3种兔球虫18S rDNA片段,产物纯化后测序。将3种球虫18S rDNA测序结果与GenBank中发布的兔球虫18S rDNA序列用DNAStar软件进行比对。使用MEGA4.0软件对兔球虫18S rDNA进行同源性比较,并绘制遗传进化树。结果表明,大型艾美耳球虫扩增出大小为1 521bp的18S rDNA片段;黄艾美耳球虫及肠艾美耳球虫均扩增出大小为1 520bp的18S rDNA片段。序列比对结果显示,3种河北株兔球虫与GenBank中相应的3种兔球虫18S rD-NA(EF694016、EF694011、EF694012)相似性分别为99.6%、99.6%和100%。3种河北株兔球虫序列和GenBank中兔球虫18S rDNA序列(EF694007-EF694017)位于一个单系集群。

To further determine E.magna,E.flavescens and E.intestinalis,three species of rabbit coccidia were isolated from rabbits in Hebei and purified.Genomic DNA were extracted from their sporulated oocysts by the method of CATB.Using conservative primer of 18S rDNA of Eimeria,18S rDNA gene fragment of three spccies of rabbit coccidia were amplified and sequenced,then they was analysed by DNAStar and aligned with corresponding sequence of the other eleven species of rabbit-infecting Eimeria in the GenBank,the phylogenetic tree was obtained using MEGA4.0.The results indicated that the gene fragment of E.magna,E.flavescens and E.intestinalis was respectively amplified with 1 520,1 520 and 1 521 bp.Sequence alignment showed that percentage similarity displayed 99.6%（E.magna in GenBank vs E.magna HB）,99.6%（E.flavescens in GenBank vs E.flavescens HB）,100%（E.intestinalis in GenBank vs E.intestinalis HB）,three species of rabbit coccidia from Hebei and eleven species of rabbit-infecting Eimeria in the GenBank were located in a monophyletic cluster.