Prokaryotic Expression 、Antisense Expression And Subcellular Localization Of DsMAPK From Dunaliella Salina

Dunaliella salina,who has no cell wall, is a natural protoplast.Due to the strong Na Cl tolerance ability, simple cellular structure and convenient culture conditions,making it become an ideal pattern creature to research plants Na Cl tolerance michanism. Mitogen- activated protein kinases belong to the serine/threonine(Ser/Thr) protein kinases,they exist in all eukaryotes and play an important role in physiological processes such as plant growth regulation and resistance to adversity.Therefore,It will have important scientific significance to further explore the function of Ds MAPK gene in clarifing the highly complex molecular mechanism of Dunaliella salina cells.In this paper, on the basis of gene Ds MAPK that our laboratory has already cloned, carry out the research of Ds MAPK prokaryotic expression、antisense expression and subcellular localization.The research methods and results of this experiment are as follows:1. The open reading frame(ORF) of Ds MAPK has been amplified by PCR, and clone to the prokaryotic expression vector p GS-21a(with GST label), construct the recombinant expression vector p GS-21a-MAPK successfully. Transform the prokaryotic expression vector p GS-21a-MAPK into E. coli. BL21(DE3), the fusion protein has been expressed successfully in E. coli. BL21(DE3) after IPTG induction.Detecte the expression form of the fusion protein by SDS-PAGE, they are in the supernatant and precipitation. After purified by GST-SefinoseTM Kit,we got the high purity of purified soluble fusion protein.Western blot showed that the fusion protein can be special recognized by GST monoclonal antibody, which preliminary proved that we get the purified GST-MAPK protein.2. Reverse insert the open reading frame of Ds MAPK gene into 35 S promoter downstream of plant expression vector, construct antisense expression vector successfully.Transform it into Dunaliella salina by using Li Ac/PEG mediated method. We got the corresponding transgenic Dunaliella salina by chloramphenicol resistance and analysed Ds MAPK expression level changes of the transgenic Dunaliella salina under high Na Cl stress at different times by semi-quantitative RT-PCR. The result showed that expression of Ds MAPK gene had been effectively inhibited in transcription level.3. Transform p MDCMGN-Cat into GV3101 competent cells,then screening positive monoclonal.Infect onion epidermal cells by Agrobacterium mediated method. Observe the instantaneous expression of Ds MAPK through fluorescence microscope.The result showed that the fusion protein GFP-Ds MAPK expressed successfully in onion epidermal cells, and mainly distributed in the cell nucleus and cell plasma.