Molecular Cloning And Characterization Of A Novel MAP Kinase Gene CbMAPK3 In Chorispora Bungeana

To survive biotic and abiotic stresses, plants have developed elaborate mechanisms to perceive external signals and adjust metabolic pathways by modulating the expression of genes. Activation and/ or inactivation of appropriate genes in response to particular stimuli are mediated through well-tuned signal transduction systems. The phosphorylation cascades including mitogen-activated protein kinases (MAPKs), MAPK kinases (MAPKKs), and MAPKK kinases (MAPKKKs) have been reported to function in various signal transduction pathways from yeasts to vertebrates. In plants, a variety of genes encoding MAPKs have been identified. Numerous studies shown that plant MAPK cascades are activated by hormones, abiotic stresses, pathogens and pathogen-derived elicitors, and also activated at specific stages during the cell cycle.Chorispora bungeana fisch. & C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly capable of resisting freezing environment. Since it is a Stress-tolerant plant, we have isolated from chorispora bungeana a new MAPK cDNA CbMAPK3 and investigated the participation of CbMAPK3 as possible mediators of abiotic stresses by RT-PCR and Western blotting. We got results as below:(1) Cloning and sequence analysis of the full-length cDNA of CbMAPK3. Based on cDNA sequences of the conserved regions of plant MAPK genes, we have isolated from chorispora bungeana a new MAPK cDNA CbMAPK3. The cloned full-length cDNA of CbMAPK3 was 1419bp with a polyA tail of 15bp. The cDNA contained a 1110bp ORF encoding a protein of 369 amino acids with a calculated molecular weight of about 42.5kDa and with a PI of 5.79. A 5' untranslated region (UTR) of 91bp was found upstream of the first ATG codon, and a 3' untranslated region (UTR) of 218bp was found downstream from the stop codon. The protein exhibit closest homology to a subgroup of plant MAPKs containing Arabidopsis AtMPK3 and we thus refer to them as CbMAPK3 (genbank accession number: AY805424). The CbMAPK3 protein contained all 11 conserved amino acid and peptide motifs characteristic of 11 subdomains of protein kinases with serin/threonine specificity. The TEY motif, whichincludes the threonin and tyrosine residues whose phosphorylation is necessary for MAP kinase activation and is a characteristic feature of MAP kinase, is also conserved in the CbMAPK3 protein sequence.(2) Based on the phylogenetic tree of cloned plant MAPKs, CbMAPK3 can be grouped intosubgroup A. Comparison of the predicted protein sequences of the CbMAPK3 with MAP kinases of other plants shows that CbMAPK3 is most homologous to the Arabidopsis AtMPK3 (95%), Nicotiana tabacum NtWIPK (82%), Capsicum annuum CaMPKl (81%), PsMAPK3 (80%), Medicago saliva MsMMK4 (80%), Petrosecinum crispum PARSLEY MAPK (80%). The secondary structure of the putative CbMAPK3 protein was analyzed by SOPMA. The result showed that the putative CbMAPK3 peptide contained 46% alpha helix, 13% extended strand, 6% beta turn, and 35% random coil. The alpha helix and random coil constituted interlaced domination of the main part of the secondary structure. From the above sequence analyses, CbMAPK3 was found to have many characteristics common to the MAPKs in plant and family A members tend to share highly similar sequences.(3) Investigate CbMAPK3 expression pattern in various tissues of chorispora bungeana,the result shown that its expression has almost no tissue specificity.(4) CbMAPK3 expression patterns under cold and other abiotic stresses were analyzed bysemi-quantitative RT-PCR. After exposure to 4°C, transcript levels of CbMAPK3 increased rapidly, maximum levels was observed 0.5 h after cold treatment, then, the CbMAPK3 transcript declined gradually and fell to the basal level at 24 h. When chorispora bungeana was exposure to -4°C, transcript levels of CbMAPK3 also increased rapidly, but the transcript maintained at a high levels up to 24h. To our knowledge, this is the first description of a plant MAPK gene whose expression is inducible at -4°C treatment. The expression of CbMAPK3 could also be induced by salt stress and ABA treatment, under salt stress, the transcripts of CbMAPK3 increased to a high level after 2h and maintained high levels at 48h after treatment. When treated with ABA, the transcripts of CbMAPK3 increased significantly after 30*min, and decreased after 12h. indicating that the CbMAPK3 gene was ABA-dependent.On the whole, the CbMAPK3 transcript levels were increased by cold (4°C and -4°C), salt and ABA treatment. The increase in expression of MAPK gene may contribute to the stress response by increasing the amount of proteins available for activation. Therefore, it can be concluded that MAPK not only become activated by posttranslational phosphorylation of the highly conserved threonine and tyrosine residue through upstream kinases, transcriptional control is also an important mechanism in MAPK signaling cascades in plant.(5) Investigate CbMAPK3 protein level under cold and other abiotic stresses. Immunoblot analysis using a -C-MPK3 antibody show that the protein level of CbMAPK3 was increased significantly and maximum levels was observed at 3h after 4°C cold treatment, then, the CbMAPK3 protein level declined gradually and fell to the basal level at 24 h . but the protein level of CbMAPK3 was increased slightly by 0°C cold treatment. When treat with 150mM NaCL, the protein level of CbMAPK3 increased after 3h and declined after 12h. The increase in protein level of MAPK may also contribute to the stress response by increasing the amount of proteins available for activation. Therefore, it can be concluded that protein level control is also an important mechanism in MAPK signaling cascades in plant.(6) To comfirm whether CbMAPK3 has the osmo-protection function, the coding regionwas subcloned into prokaryotic expression vector. E. coli (sri.TnlO) cell transformed with this plasmid exhibitad no significantly better growth in the presence of 1M sorbitol, compared to E. coli (srl::TnlO) transformed with the vector only, after induction with IPTG. we can conclude that CbMAPK3 is likely involved in abiotic stress responses as a signal transduction element, but itself did not has the function of osmo-protection.(7) Expression of CbMAPK3 in E. coli BL21. The coding region of CbMAPK3 cDNA wassubcloned into the bacterial expression vector pET30a, and transformed E. coli BL21. After induced by IPTG, the expression protein had a molecular mass of about 46kD by SDS-PAGE, corresponding to the deduced amino acid sequence of CbMAPK3 fusion protein. Western blotting analysis using a -C-MPK3 antibody showed that the BL21/ pET30a/CbMAPK3 samples had positive signal at 46kD position, where there is nosignal in BL21/ pET30a samples. This result confirmed that a -C-MPK3 antibody specifically reacted with CbMAPK3 protein. So, we can further conclude that the protein reacted with a -C-MPK3 antibody in the whole protein of Chorispora bungeana is CbMAPK3.In conclusion, a novel MAPK gene CbMAPK3 was identified and characterized. The expression of CbMAPK3 was induced by cold, salt, and ABA but no tissue specificity.The protein level of CbMAPK3 increased when treated with cold (4 and -4 °C) and salinity stress. These results indicate that the CbMAPK3 may play an important role in response to environmental stresses.