A Novel Rabl-like Gene with A 25 bp Intron in Trichomonas vaginalis / 热带医学杂志

ABSTRACT

Objective The aim of this study is to clone and characterize a novel Trichomonas vaginalis Rabl-like geue (TvRabl-like) with a small intron. Methods The eDNA clone of TvRabl-like gene was isolated from a cDNA expression library and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST ClustalW programs.Phylogenetic analysis was carried out by MEGA3 program. The genomic DNA and mRNA of TvRab1-like gene were amplified using PCR and RT-PCR techniques respectively and also sequenced. Results The eDNA sequence of TvRab1-like gene had a length of 705 base pairs with an open reading frame of 603 bp. The deduced amino acid sequence from the open reading frame possessed 200 residuals corresponding to a putative M.W. 22532.2 and an estimated pl of 7.4. Sequence analysis demonstrated that TvRab1-like gene showed the highest homology to T. vaginalis Rabla (63% identity and 79% similarity) and the Rabl subfamily of other species, suggesting that the deduced amino acid sequence from this cDNA clone was a Rabl isoform. Phylogenetic analysis showed that TvRab1-like gene was clustered with T.vaginalis Rab1 subfamily in the phylogenetic tree. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA possessed a 25 bp intron which contained canonical 5' GT-AG-3' and branch site motifs as those larger introns found in T.vaginalis and other eukaryotes. The analysis of RT-PCR products demonstrated the presence of the unspliced mRNA and spliced mRNA, indicating that there was a intron. Conclusion These data suggest that TvRabl-like gene belongs to T.vaginalis Rabl subfamily. TvRabl-like gene possesses a 25 bp splieeosomal intron which is the smallest one of the introns identified in this deepest-branching protist and might be the shortest intron of eukaryotes. Study of those introns might provide more insights into the intron evolution of eukaryotes.