Construction Of Recombinant Vector And Prokaryotic Expression And Subcellular Localization Of Gene DsSTPK From Dunaliella Salina

As we know,Dunaliella salina are lower eukaryotes which can able to withstand high salt,so it became the model organism for the study of how plants adapt to high salt. Over the years,many scholars studied how the osmotic adjustment to adapt to high salt environment, cloning ofthe salt-related genes, membrane and cytoplasmic proteins, etc. from salina, has made someprogress.But as so far, the molecular mechanisms and signal transduction pathways remainunclear. Phosphorylated proteins is a major pathway for cells to respond to various externalstimuli. Protein kinase is a key signaling molecule which plays an important role in signaltransduction by phosphorylatting the substrate. Serine/threonine protein kinase (STPK) is oneof the most important class of protein kinases, can catalyze a variety of function protein’sphosphorylation (such as enzymes, receptors, transport proteins, regulatory proteins, nuclearproteins, etc.), which regulate many cellular processes of life. Therefore,the research of serine/threonine protein kinase has important scientific significance to elucidate the mechanism ofcomplex salt tolerant in algae.This study constructed prokaryotic expression vector pGS-21a-DsSTPK, eukaryoticexpression vector pMDCSGN and pBI121-DsSTPK, finished the prokaryotic expression andsubcellular localization of the DsSTPK gene from Dunaliella salina, provided new informationfor further research of the molecular mechanisms about how STPK works. The mainexperimental methods and results are as follows:1. Amplified DsSTPK gene by PCR, and cloned into the pMDTM18-T Simple Vector. DNAsequencing results showed that the nucleotide sequence is not mutated. The cloning vector andexpression vector were digested by restriction endonuclease, then the target fragment andvector’s large fragment were linked by T4DNA ligase, so the prokaryotic expression vectorpGS-21a-DsSTPK,eukaryotic expression vector pMDCSGN and pBI121-DsSTPK wereconstructed successfully, make sure the reading frame were correct by sequencing.2. The prokaryotic expression vector pGS-21a-DsSTPK was transferred into E. coli. BL21(DE3), and expressed successfully after the inducing of IPTG. Detected by SDS-PAGE, thefusion protein were present both in the supernatant and inclusion bodies. After been purified byGST-tagged protein purification,we obtained a high purity soluble fusion protein, Western blotshowed that the fusion protein can be recognized specifically by anti-GST monoclonalantibody,it showed the protein P-GST-DsSTPK were purified successfully.3. The eukaryotic expression vector pMDCSGN was transferred into into Agrobacterium LBA4404competent cells, then filtered by Ampicillin. Transfred it into onion epidermalcells by the Agrobacterium media, observed the expression of DsSTPK under a fluorescencemicroscope. The results showed that the fusion protein GFP-DsSTPK were expressed both inonion epidermal cell membrane and cytoplasm. Proved in Dunaliella salina the DsSTPK proteindistributs both in the cell membrane and cytoplasm.