Cloning And Expression Of DsMAPK From Dunaliella Salina

Dunaliella salina is an important model organism for investigating salt tolerancemechanism of plant, and MAPK is the key gene in the molecular pathway of plant salt tolerance.In this experiment the full length cDNA of MAPK gene from Dunaliella salina named asDsMAPK was cloned using RT-PCR and RACE technology.The gene expression patterns undersalt exposure were analyzed using Real time PCR, and eukaryotic expression vector forDsMAPK gene was constructed using LiAc/PEG-mediated method. The results in this study willbe helpful for investigating the molecular response to salt stress in Dunaliella salina.1.Partial sequence of DsMAPK was amplified using degenerate primers, and the full lengthcDNA of this gene was obtained using SMART RACE technology and specific primers designedby target sequence. The full length of the target gene was1861bp containing67bp5′-UTR,387bp3′-UTR and1407bp ORF encoding468amino acids. The gene was named DsMAPKand the GenBank Accession number was JQ782412.2.Sequence analysis revealed that the formula of the instable hydrophilic protein encodedby DsMAPK gene was C2286H3593N641O706S20, with a total atom number of7246, a relativemolecular weight of52kDa and aisoelectric point of5.87. The protein was a non-transmembrane protein without signal peptide, located in cytoplasmic matrix. The secondarystructure prediction showed that α-helices and free curl were the mainly structural elements ofthe protein secondary structure and extended chains scattered among them. Amino acid sequencehomology analysis implied that DsMAPK was closer to the MAPK protein of Chlamydomonasand Volvox.3.This study analyzed the expression patterns of DsMAPK gene response to salt stress byQRT-PCR. The result showed that the expression of DsMAPK achieved the highest level and wasfive fold to the control group after1h stimulation (P<0.01). Expression of DsMAPK gene wassignificantly upregulated by salt change, which indicated that DsMAPK gene may participate inthe resistance to salt stress.4.Green fluorescent protein (GFP) including restrict digestion sites of Xbal I and Sal I wasamplified using plasmid as a template. The amplification product was ligated into pMDCKN-Catvector to construct expression vector pMDCKN-Cat. The cDNA of D.salina was used astemplate to amplify the ORF of DsMAPK gene (including restrict digestion sites of BamH I andXbal I). The amplification product was ligated to the expression vector pMDCKGN-Cat toconstruct the eukaryotic expression vector DsMAPK. The eukaryotic expression vector wastransformed into the cells of Dunaliella employing LiAc/PEG-mediated method under theoptimal conditions (transformation temperature29℃, vector density600μg/ml, Dunaliellacultured for5days). Transient expression of the fusion protein of MAPK and GFP was observed by using fluorescent microscope.