Cloning And Expression Analysis Of Serine/threonine Protein Kinase Gene DsSTPK From Dunaliella Salina

Dunaliella salina is a kind of unicellular eukaryotes, belonging to Dunaliella,Dunaliellaceae, Chlamydomonadales, Chlorophyceae, Chlorophyta, is known as one of the mosthalotolerant eukaryotes, which is a model organism for the research of plant tolerance to saltstress. Over the years, studies on the aspects of the osmoregulation of Dunaliella salina in thehigh-salt environment, cloning of salt tolerant related genes, the plasmamembrane and cytosolicproteome of Dunaliella salina have been carried out by many scholars. Although some progresshave been achieved, so far the molecular mechanisms and signal transduction pathways ofDunaliella salina tolerance to salt stress remain unclear.Protein phosphorylation is an important mechanism for the regulation of cell response tovarious exogenous signals. Protein kinase plays an important role in cell signal transduction byphosphorylating substrate proteins. Cellular basic processes such as cycle regulation, growth anddevelopment, division and differentiation are regulated by specific protein kinase. Theserine/threonine protein kinase （STPK） is the most important category of protein kinase, whichregulate a variety of biological processes by catalyzing the phosphorylation of functionalproteins （such as enzymes, receptors, transport proteins, regulatory proteins, nuclear proteins,etc.）. The study of serine/threonine protein kinase has an important scientific significance inclarifying the molecular mechanism of tolerance to salt stress. Therefore, in this article, weisolated and characterized the serine/threonine protein kinase gene from Dunaliella salina, inaddition, we also investigated its expression. The main experiment methods and conclusions ofthe thesis are as fellows:1. A serine/threonine protein kinase gene （GenBank Accession No.JN625213, designatedDsSTPK） was isolated from halotolerant alga Dunaliella salina using RT-PCR and rapidamplification of cDNA ends （RACE） method. The full-length cDNA was3129bp, containing a242bp5’-untranslated region （UTR）, a703bp3’-UTR, and a2184bp complete open readingframe （ORF） encoding a polypeptide of727amino acids.2. Bioinformatics analysis showed that DsSTPK was a hydrophilic protein which had notransmembrane domains and signal peptide, located in the cytoplasmic matrix; DsSTPKcontained two conserved regions （a protein kinases ATP-binding region in167-175and aserine/threonine protein kinases active-site in293-305） and existed some potentialphosphorylation sites; The main element of the secondary structure are random coil and α-helix,the catalytic domain in the three-dimensional structure to form an active cavity; The amino acidsequence shared the highest homology with the serine/threonine protein kinase of Chlamydomonas reinhardtii （65%identities, XP₀01701465.1）, Phylogenetic analysis indicatedthat DsSTPK had the closest genetic relationship with the serine/threonine protein kinase ofChlamydomonas reinhardtii and Volvox carteri; Activity analysis showed that the binding energybetween DsSTPK and activator phosphatidylserine （PS）、diacylglycerol （DAG）、phorbol ester（TPA） were lower, and catalytic histone serine/threonine residues phosphorylated.3. We investigated the expression pattern of DsSTPK at the transcription level in response tosalt stress （3M） using the real-time quantitative PCR （QRT-PCR） method. The results showedthat the expression level was significantly up-regulated and reached the peak level at12h after3M NaCl treatment, which increased130%than that in normal growth conditions （P<0.05）. Astime progressed, the expression level decreased and recovered to the control level.4. Using recombinant DNA technology, the prokaryotic expression vectorpET32a（+）-DsSTPK was constructed by connecting the open reading frame of DsSTPK and theplasmid pET32a（+）. The expression vector pET32a（+）-DsSTPK was transformed into E. coliBL21（DE3）, and then induction with isopropyl-β-D-thiogalacropyranoside （IPTG）. The fusionprotein existed in both soluble and inclusion body. The high purity soluble fusion protein wasacquired by purified supernatant protein. Western blot identification confirmed that the purifiedfusion protein can be specifically recognized by anti-his antibody.These results of this study not only provide a new information to further explore themolecular mechanism of Dunaliella salina adapt to the high salt environment, but also laid afoundation for the next step to study the role of DsSTPK in salt tolerance mechanism ofDunaliella salina at the protein level.