我国羊巴贝斯虫trap基因结构及遗传进化分析

The Structural and Phylogenetic Analysis of trap Gene in Ovine Babesia Species in China

本研究拟解析我国羊巴贝斯虫trap基因的结构特征,并以其为分子标志开展遗传进化分析,为理清我国羊巴贝斯虫的分类关系提供数据。用公布的巴贝斯虫trap基因序列BLAST两个羊巴贝斯虫全基因组本地数据库,以获得的保守序列片段为模板,设计PCR引物;克隆我国分离的6株羊巴贝斯虫trap基因全长序列,分析其序列结构特征;比对序列相似性,构建系统发生树,分析其遗传进化关系。结果克隆获得6株羊巴贝斯虫trap基因,分析结果显示,其具有3、4、5个内含子的三种内含子数目,预测的开放阅读框(ORF)大小分别为1 944、2 100/2 142和2 286bp;氨基酸序列分析显示,其都具有TRAP蛋白家族特征性的vWFA、TSR、跨膜域和CTD结构域;进化关系分析显示,感染我国羊的巴贝斯虫被分成3组,组内序列相似性为99.8%~100%,而组间为43.6%~74.6%。系统发生树上6株羊巴贝斯虫被分到两大枝,羊巴贝斯虫未定种和莫氏巴贝斯虫,而且莫氏巴贝斯虫又被分成两小枝。6株羊巴贝斯虫trap基因结构存在差异,但其蛋白特征性的结构域较为保守;我国分离的羊巴贝斯虫为两个种,但莫氏巴贝斯虫中可能存在两个亚种。

This study focused on the structural characteristics of Chinese ovine Babesia trap gene via sequencing the full-length sequence and bioinformatics analysis.And then a phylogenetic analysis was performed on the TRAP nucleotide and amino acid sequences,which provided the data for clarifying the taxonomic relationship of Chinese ovine Babesia.The conserved sequence fragments were obtained using published Babesia trap gene sequences to BLAST 2of local genomic databases of Chinese ovine Babesiastrains.The primers of PCR were designed with the fragment sequences as templates.The full-length trapgene sequences of 6Chinese ovine Babesia strains were cloned and their structural characteristics were analyzed.The evolutionary relationship of these ovine Babesiastrains was analyzed based on the comparisons of TRAP sequence similarities and constructed phylogenetic trees.The full-length gene sequences of trap were cloned from 6ovine Babesiastrains.The results of structural characteristics analysis showed that the trapgene of 6ovine Babesiastrains had 3patterns of intron,3,4and 5introns and the predicted ORF sizes were 1 944,2 100/2 142 and 2 286 bp,respectively.The analysis of amino acid sequences indica-ted that the predicted ovine Babesia TRAPs possessed all the distinctive domains of TRAP protein family,such as vWFA,TSR,transmembrane motif and CTD.Phylogenetic analysis showed that these Chinese ovine Babesiastrains could be divided into 3groups.The similarities of intragroup between the TRAP sequences were between 99.8% and 100%,and those of inter-group were from 43.6% to 74.6%.The 6ovine Chinese Babesia strains were divided into 2clades,Babesiasp.and B.motasi clades.In addition,the B.motasi clade were further divided into 2sub-clades.The trapgene structure of 6ovine Babesia strains are various,but the distinctive protein domains are conserved.These Chinese ovine Babesiastrains belong to 2 Babesiaspecies,and possibly there are 2sub-species in the B.motasi species.