Construction Of Recombinant Vector, Prokaryotic Expression And Sub-cellular Localization Of Dsrab From Dunaliella Salina

Dunaliella salina is a kind of unicellular algae. Salina without cell wall is aprotoplasts coated by only one membrane. Currently, salina with the mostsalt-tolerance,can be used to study salinity tolerance mechanism. It have beenrevealed that, salina adapts to short-term salt stress mainly by ion pump regulationand glycerol synthesis. Under high salt stress for prolong time, many specific processof salinity tolerance mechanism have to be dissected,for instance,how to induce thegenes associated with the salt-tolerance,and how to activate the tolerant signalingpathway. Rab proteins are the largest subfamily in small GTP binding protein family.Rab, as the molecular switch, play a significant role in cell physiologicalactivities,such as the cytoskeleton reorganization, vesicle transportation, cellproliferation, signal transduction. In recent years,many Rab proteins associated withsalt tolerance have been found in multiple organisms. Studying on DsRab protein, isimportant for revealing the molecular mechanism of the salina response to salt stressprocess. Methods and results of this study are as follows:1.The open reading frame (ORF) of DsRab gene was cloned in prokaryoticexpression vector pGS-21a to construct the recombinant plasmid pGS-21a-DsRab.The ORF DsRab was connected with pMDCMGN to construct sub-cellularlocalization recombinant plasmid pMDCGGN. The eukaryotes expressionrecombinant plasmid pBI121-DsRab was constructed by connecting the ORF DsRaband the plasmid pBI121.2. The recombinant plasmid pGS-21a-DsRab was transformed into E. coli. BL21(DE3). The recombinant protein was expressed in suspernatant form and inclusionbody.To harvested more supernatant recombinant protein, the prokaryotic expressioncondition was optiminzed,and the optimal conditions is28℃，0.01mol/L IPTG. Theproteins were purified by GST gravity-flow columns, then identified by Western blot.3. The sub-cellular localization recombinant plasmid pMDCGGN wastransformed into Agrobacterium tumefaciens LBA4404. The onion epidermal cellswere transfected by Agrobacterium tumefaciens-mediated method. The recombinantprotern GFP-DsRab were expressed successfully in onion epidermal cells and mostlylocated on membrane.This study have constructed successfully recombinant vectors:pGS-21a-DsRab,pMDCGGN and pBI121-DsRab.A large quantity of highly purified recombinantprotern GST-DsRab were acquired with the prokaryotic expression method. TheGFP-DsRab were mostly located on membrane of onion epidermal cells. These results not only preparat for the next step to study the DsRab,then selecting the interactioninprotein with the DsRab,but also laid a foundation for explore the effect of DsRab intransgenic plants adapt to the high salt environment.