Isolation And Function Identification Of A Novel Mitogen-Activited Protein Kinase Gene, MhMAPK, In Malus Hupehensis Rehd. And Its Role In Plant Signal Transduction

Mitogen activated protein kinases (MAPK) are key enzymes in Mitogen Activated Protein Kinase (MAPK) cascades which are common signal transduction pathway in the process of growth, development, biotic and abiotic stress responses. Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang(Chinese name is PingYi TianCha, PYTC) is the common rootstock of malus.In this thesis, a series of studies based on the materials of Malus hupehensis (Pamp) Rehd. var pinyiensis Jiang and have been conducted on the gene isolation, sequence comparison, expression analysis, kinase activity analysis under stress. The main results are as follows:1. Isolation and characterization of ZmOSMAPK1 geneAccording to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from PYTC roots. By RT-PCR and RACE-PCR, we isolated a MAPK gene from PYTC roots and named MhMAPK, with the accession number of GeneBank EF427897.2. The software analysis of MhMAPKSoftware analysis showed that MhMAPK protein was a hydrophobic protein, which contained two speculated transmembrane(TM) segments. The 217-235aa TM segment has a strong transmembrane preference from inside to outside. Sequence comparison showed that the MhMAPK protein was closer to AtMPK4 of Arabidopsis and NtMPK4 of tobacco. Similar to other MAPK proteins, MhMAPK contained all 11 conserved subdomains, a putative phosphorylation TEY motif, and a common docking domain (CD domain) in its C-terminal extension. These results indicated that MhMAPK was a homolog of AtMPK4 and NtMPK4 and belonged to C group MAPKs. 3.The subcellular localization of MhMAPKWe constructed the vector of pBIN-GFP-MhMAPK, which then was introduced into onion inner epidermis, the result showed that MhMAPK localized in the nucleus, and the software ProtComp Version 6.1 also indicated that MhMAPK localized in the nucleus. Maybe MhMAPK play a role in the phosphorylation of nuclear factors.4. Construction of expression vectorConstruction vectors pBI121-MhMAPK containing full-length cDNA of MhMAPK gene in sense and pBI121-3'segment containing 3'segment in antisence orientation driven by the constitutive cauliflower mosaic virus 35S promoter were assembled and introduced into tobacco, and gained transgene plants in sense.5. The response of MhMAPK to the stress in Malus hupehensis (Pamp) Rehd. var pinyiensis JiangAccording to the analysis of Real time PCR and MAPK activity, the results showed that the MhMAPK gene expression increased under the high salinity stress, drought stress, CdCl2 and CuCl2 stress and SNP treatment. SNP, CdCl2 and CuCl2 were all able to induce the increase of MAPK activity. Besides, CdCl2 and CuCl2 could all induce the generation of nitric oxide and 100μM SNP could decrease the ROS content and increase the activity of SOD, POD significantly. These results showed that nitric oxide might play a role in the Cd and Cu signal transduction as a messenger molecule and activated MAPK cascades might participate in the Cu, Cd response and nitric oxide signal transduction.