The Diversity Of Rab Gene Sequences And Characterization Of An Atypical Rab Protein In Euplotes Octocarinatus

Rab proteins are a family of small GTPase and expressed in all eukaryotic cells.The different Rabs bind different effectors and have unique subcellular localizations to the cytosolic face of specific intracellular membranes,where they function as regulators of distinct steps in membrane traffic pathways.There are some researches on the diversity of Rab genes in order to explore the vesicular transport network in eukaryotic cells.Ciliate,a unicellular protozoa and low-class eukaryotes in evolution,has perfect vesicular trafficking system among organelles.Identification and characterization of the Rab genes from ciliate would facilitate to understand the mechanism of vesicular transportation.To further the study of diversity and function of Rab proteins in evolution,Euplotes octocarinatus,a kind of unicellular protozoa ciliate,was used as material in my thesis.Rab genes were screened by degenerated primer PCR method from macronuclear DNA of Euplotes octocarinatus,and 11 novel Rab genes were obtained.According to the systematic nomenclature based on their homologies,we named them as EoRab1Z,EoRab2a, EoRab2b,EoRab2c,EoRab2d,EoRab6,EoRab7,EoRab43,EoRab2-like, EoRabL2,and EoRan,respectively.Among these EoRabs,EoRab43 is an atypical Rab gene for it cannot be classified into any Rab subfamily which had been identified.Being the first atypical Rab gene identified in Euplotes octocarinatus,EoRab43 is assumed to play some special functions in this ciliate on the basis of sequence analysis. In order to understand the function of EoRab43,the 153bp nucleotides of the 3'-end of EoRab43 gene was cloned into expression vector pGEX-6P-1,and the recombinant vector pGEX-6P-1-EoRab43_153bp was transferred into E.coli BL21(DE3) to express the fusion protein.Western blotting assay with GST antibody confirmed that the protein is GST-EoRab43_50aa. The fusion protein GST-EoRab43_50aa was purified by affinity chromatography.BALB/c mice were immunolized by purified GST-EoRab43_50aa.The titer of anti-EoRab43_50aa polyclonal antibody was detected by indirect ELISA assay and the specificity of the antibody was detected by Western blotting.The potency of the antibody was as high as 1:60000.The obtained anti-EoRab43_50aa polyclonal antibody was used for localization of EoRab43 protein in Euplotes octocarinatus. Immunofluorescence experiments were performed using anti-EoRab43_50aa antibody in the cells of Euplotes.The results showed that EoRab43 displayed a punctuate pattern in the cytoplasm around the macronuclear chromosome of Euplotes.