Isolation And Characterization Of A Mitogen-activated Protein Kinase Gene In Halotolerant Algae Dunaliella Salina

Mitogen-activated protein kinase (MAPK) is conserved gene family in all eukaryotic cells. MAPKs play an important role in abiotic stress responses. In green algae, a few of MAPK genes have been cloned, although MAPKs have been extensively studied in yeasts and humans. The microalga Dunaliella is a model in study of variety of abiotic stress tolerances. Dunaliella salina performs excellent adaptation to the hypersaline environment whithout rigid cell wall. Each MAPK pathway contains a threetiered kinase cascade comprising a MAP kinase kinase kinase (MAPKKK, MAP3K, MEKK or MKKK), a MAP kinase kinase (MAPKK, MAP2K, MEK or MKK) and the MAPK. Receptor mediated activation of a MAPKKK. MAPKKKs are serine/threonine kinases that activate MAPKKs through phosphorylation on two serine/threonine residues. MAPKKs are dual-specificity kinases that phosphorylate MAPKs on threonine and tyrosine residues in the TxY motif. MAPKs are promiscuous serine/threonine kinases that phosphorylate a variety of substrates. This threetier module mediates ultrasensitive switchlike responses to stimuli. The assembled sequence of DsMPK was 1849 bp in length. The cDNA contained a 193 bp 5'-UTR, a complete ORF encoding 472 amino acids followed by 3'-UTR of 237 bp. The DsMPK shared a homology of 57.4 to the MAPKs from plant, yeast and animal at the amino acid level. Blast analysis showed that the deduced DsMPK amino acid sequence had a high level of similarity to plant MAPK kinases (74% CrMPK; 60% AtMPK4; 63% AtMPK6; 62% AtMPK3). The conserved regions, including TEY activation loop and the common docking domain, were characterized in the deduced amino acids sequence.Three fragments (1.5kb, 1kb, 700kb) was amplified from D. salina genome DNA, The assembled sequence of DsMPK was 2727 bp in length. The sequence contains two introns. The analysis of GC% finds that the GC percentage of exon is higher than intron. The second intron follows the GT-AG rule. Interestingly, the first two exons of the DsMPK gemome DNA are short, but the third is longer and possess the major function domain. It propose that the first two exon and the first intron are mediator. The Southern blotting shows that there is only one copy of the DsMPK genome DNA.The expressions of DsMPK gene under hyperosmotic and low temperature condition were examined at fixed time, using QRT-PCR. The results showed that under conditions of hyperosmotic stress expression of the DsMPK gene is reduced. The expression of DsMPK reduced within 5 min under hyperosmotic stress. After 30 min, the expression of DsMPK decreased to 50 percent. Minimum expression of DsMPK gene was reached at about 6 h. Under low temperature condition, the expression of DsMPK gene rapidly reduced as indicated by the QRT-PCR. With in 5 min, expression decreased by 50 percent. After gradual decrease for 6 h, the expression of DsMPK reached its minimum level. Therefore, the role of DsMPK in stress signaling transduction is uncertain, because MAPK can be regulated at the level of transcription, translation, and/or post-translation. The translation and post-translation changes would be detected by Western blotting and kinase assay.Therefore, the DsMPK is constructed into prokaryotic expression vector pET-32a, and induced to express in E. coli BL21. Target protein is purified with affinity chromatograph, which prepare for the further research.