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目的克隆和鉴定一个新的阴道毛滴虫 Rab1-like 基因( TvRab1-like) 及其内含子。方法我们从一阴道毛滴虫 cDNA 表达文库中分离出一个 cDNA 克隆, 它与各物种的 Rab 家族蛋白有较高的同源性, 因此我们进一步用 BLASTP、RPS-BLAST、ClustalW 和 MEGA3 等分析软件对该 cDNA 克隆进行了序列分析和进化树分析; 用 PCR 和 RT-PCR 等技术分别对该基因组和 mRNA 进行了扩增和测序分析。结果序列分析结果表明该cDNA 克隆长 705 bp, 开放阅读框具 603 bp, 推测肽链含有 200 个氨基酸。序列比较分析结果提示该 cDNA 克隆所推测的蛋白质是一个 Rab1 亚家族的亚型。进化树分析也表明它属于阴道毛滴虫 Rab1 亚家族。基因组 PCR扩增和测序分析表明该基因包含一个 25 bp 的内含子, 该内含子具有阴道毛滴虫和其它真核生物较大内含子所具备的典型的 5′GT-AG-3′和分支位点基序。RT-PCR 产物及其测序分析表明在该基因的转录本中存在着未剪切和剪切后的 mRNA, 说明确实有内含子的存在。结论 TvRab1-like 基因属于阴道毛滴虫 Rab1 亚家族, 该基因含有一个 25 bp 的内含子。该内含子是至今发现的最小的阴道毛滴虫基因内含子之一, 很可能也是真核生物中最小的内含子。对诸类最低等真核生物内含子的研究将有助于我们理解真核生物内含子的起源和进化。
Objective To clone and identify a novel Rab1-like gene (TvRab1-like) and its introns. Methods We isolated a cDNA clone from a Trichomonas vaginalis cDNA expression library, which has high homology with the Rab family of proteins of various species. Therefore, we further analyzed the protein by BLASTP, RPS-BLAST, ClustalW and MEGA3 The cDNA clone was analyzed by sequence and phylogenetic tree. The genome and mRNA were amplified and sequenced by PCR and RT-PCR, respectively. Results The sequence analysis showed that the cDNA clone was 705 bp in length and 603 bp in open reading frame. It was deduced that the peptide contained 200 amino acids. Sequence comparison analysis suggested that the protein predicted by this cDNA clone is a subtype of Rab1 subfamily. Phylogenetic tree analysis also showed that it belonged to the Trichomonas vaginalis Rab1 subfamily. Genomic PCR amplification and sequencing analysis revealed that the gene contains a 25 bp intron with the typical 5’GT-AG-3 of Trichomonas vaginalis and other larger eukaryotes ’And branching site motifs. The RT-PCR product and its sequencing analysis showed that there are uncut and cleaved mRNA in the transcript of the gene, indicating that there is indeed an intron. Conclusion TvRab1-like gene belongs to the Trichomonas vaginalis Rab1 subfamily, which contains a 25 bp intron. This intron is one of the smallest introns found in Trichomonas vaginalis to date and is probably the smallest intron in eukaryotes. Research on the lowest types of eukaryotes such as introns will help us to understand the origin and evolution of eukaryotes.