| Cellular organelles of eukaryotes have a distinctive spatial distribution and communicate through an elaborate system of vesicular-tubular transport. Rab proteins, behaving as molecular switches of vesicular trafficking, play an important role in different steps of vesicular trafficking. Ciliate, a unicellular protozoa and low eukaryotes in evolution, has perfect vesicular trafficking system among organelles. It's meaningful to study the function of Rab proteins in ciliates and further to explicit the mechanism of vesicular trafficking.In this study, a rab gene was obtained from Euplotes octocarinatus by polymerase chain reaction (PCR) and RT-PCR. The rab gene from macronucleic DNA was 884 bp in length, including non-coding regions and telomeric sequences at both ends. The rab gene from micronuclear DNA (723 bp), lacking of internal eliminated sequences, was identical to rab gene from macronuclear DNA. RT-PCR showed that the opening reading frame of the rab gene was 663 bp long. The rab gene from macronuclear DNA contained an intron of 60 bp at the position from 153 bp to 212 bp of macronuclear DNA. The rab gene had two in-frame TGAs encoding for cysteine in Euplotes octocarinatus. The rab gene used TAG as stop codon, which was the first report in Euplotes octocarinatus. The result of BLAST in NCBI demonstrates that the Rab shares a homology of 49-52 % at the amino acid level with Rabl proteins from a number of other eukaryote, which suggesting that the Rab is a Rabl homolog. The rab gene was therefore designated Eo-rab-1N.In order to understand the function of the Rab protein in ciliates, the coding region of Eo-rab-1N obtained from mRNA of Euplotes octocarinatus was ligated into plasmid PGEM-T Easy. To get the gene expressed in E. coli, two in-frame TGA condons were mutated to TGC by site-directed PCR mutagenesis. The mutated gene Eo-rab-lN was cloned into expression vector pRSETc. The expression of E.coli BL21(DE3)/pRSETc-Eo-rab-lN was...
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