柔嫩艾美耳球虫杨陵株3-1E基因的克隆与序列分析

Cloning and Sequence Analysis of 3-1E Gene ofEimeria tenellaYL Strain

根据GenBank发表的鸡柔嫩艾美耳球虫(Ei meriatenella)3-1E基因的ORF设计一对引物,用RT-PCR方法从E.tenella杨陵(YL)株总RNA中扩增3-1E基因,并将扩增产物与pMD18-T easy载体进行连接并测序,用DNAstar 5.0软件对测序结果进行分析。结果表明,2009年分离的E.tenellaYL株3-1E基因与其他虫株相比,核苷酸有9个位点发生变化,其中7个位点的变化引起了相应氨基酸位点的改变,其他2个位点的变化未引起氨基酸的改变。对2006年和2009年分离的YL株3-1E基因的核苷酸进行了比对,结果发现,有两个核苷酸位点发生了变化。系统发育树分析表明,E.tenellaYL株3-1E基因同种间与E.tenella甘肃株遗传关系最近,同属间与E.acervulina法国株遗传关系最近。

Based on the published ORF of 3-1E gene inEimeria tenella,one pair of primers was de-signed.The gene was amplified by RT-PCR fromE.tenellaYL Strain.Then the amplicon was cloned into pMD18-T easy vector and sequenced.The sequence of 3-1E gene was analyzed by DNAstar 5.0 software.Compared 3-1E gene ofE.tenellaYL strain with other strains,nine mutant nucleotide sites were detected,of which,seven nucleotide site variations led to the mutation of corresponding amino acid sequences.There were two nucleotide site variations between the YL strains isolated in 2006 and in 2009 respectively.The phylogenetic tree revealed that 3-1E gene ofE.tenellaYL strain had close relationship with that ofE.tenellaGS strain in same species,and with that of E.acervulina France strain in same genus.