Rab鸟苷三磷酸酶是细胞膜转运机制的关键调节分子,与原虫的分泌功能密切相关.本研究旨在克隆和分析阴道毛滴虫Rab6鸟苷三磷酸酶同源基因,以便进一步探讨其功能.作者从阴道毛滴虫cDNA表达文库中分离出2个Rab6鸟苷三磷酸酶同源基因的cDNA克隆,其中1个cDNA序列长658对碱基,读码框含597对碱基,推测蛋白质序列具198个氨基酸.序列分析表明该氨基酸序列与Rab6a蛋白亚家族的同源性最高.另一cDNA序列长764对碱基,读码框含657对碱基,推测蛋白质序列具218个氨基酸.序列比对分析显示该氨基酸序列与Rab6b蛋白有较高的同源性.2个氨基酸序列都拥有Rab鸟苷三磷酸酶家族的所有保守结构域、特异性RabF结构域和典型的异戊烯化结构域.进化树分析提示毛滴虫的这2个基因系Rab6家族的同源基因,在进化上更接近原虫和单细胞的酵母Rab6亚家族.序列分析还显示这2个基因都无内含子,其基因组DNA序列与其cDNA序列完全一致.%Rab GTPases are key regulators of cellular membrane trafficking which is involved in the function of secretion in protozoa. The aim of the study is to clone and characterize Rab6 genes from Trichomonas vaginalis. A cDNA expression library was constructed with T. vaginalis total RNA. Two cDNA clones, which showed high homology with Rab proteins of different species, were isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs, etc. The genomic DNA corresponding to the cDNA sequences was amplified using PCR techniques and sequenced. One of the cDNA sequences has a length of 658 base pairs with an open reading frame of 597 bp. The deduced amino acid sequence from the open reading frame possesses 198 residues. Sequence analysis demonstrated that this cDNA clone was most homologous to the Rab6a subfamily of different species. The other one has a length of 764 bp with an open reading frame of 657 bp. The deduced peptide sequence contains 218 amino acid residues. Sequence comparison showed this cDNA clone had significant similarity to Rab6b GTPases of other species. Both the deduced amino acid sequences contain all the known conserved sequence elements of Rab family, specific RabF motifs, and typical prenylation motifs as well. Phylogenetic analysis showed that the two trichomonad Rab6 are clustered with Rab6 proteins from other species, and rather close to the protozoon and yeast Rab6 sub-family. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequences encompassing the putative 5'-ATG and 3'-stop codons are identical to their cDNA sequences.These data suggest that the two Rab genes are T. vaginalis Rab6a and Rab6b homologs which possesse no intron. Their functions in regulating membrane trafficking pathways of the parasitic protist remains to be studied.
展开▼
