The Study Of Molecular Mechanism Of Mitogen-activated Protein Kinase Cascades In Regulating Stresses In Canola And Arabidopsis

Canola (oilseed rape, Brassica napus L.) is a major oil crop worldwide. Its quality andyield are often limited by adverse environmental stresses including salinity, drought, andfungal pathogens and so on. Sclerotinia sderotiorum is a phytopathogenic necrotrophic fungithat causes stem rot in many plants including canola. Therefore, it is important to mine genesresponsive to Sclerotinia sderotiorum and conferring plants tolerance to the fungal disease.Eukaryotic mitogen-activated protein kinase (MPK) signaling cascades can transduceand amplify environmental signals via three types of reversibly phosphorylated kinases tocause a series of physiological and biochemical reaction and downstream gene expression. Sofar, there is few report on identification and functional characterization of MPK and itsupstream MAPK kinases (MKK) as well as downstream WRKY transcription factors (TFs) incanola.In the previous work, we have identified and cloned seven MKKs (BnaMKK),12MPKs(BnaMPK) as well as multiple WRKY TF members from canola. In the present study, we alsoexamined the subcellular localization of two and one members of BnaMKK and BnaMPKgene families and one member of WRKY gene family, respectively, using green fluorescentprotein (GFP) and, found GFP signals in both nuclei and/or cytoplasm. Furthermore, we haveidentified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis ofinteractions between BnaMKKs and BnaMPKs, as well as BnaMPKs and BnaWRKYs. Wedefined several new contiguous signaling modules includingBnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26andBnaMKK9-BnaMPK5/9/19/20. Selected interactions were validated in vivo by bimolecularfluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canolaMKK, MPK and WRKY genes to stimuli including fungal pathogens, hormones and abioticstress treatments were analyzed through real-time RT-PCR and I have identified a few ofBnaMKKs, BnaMPKs and BnaWRKYs responding to salicylic acid (SA), oxalic acid (OA),Sclerotinia sclerotiorum or other stress conditions.From these WRKY genes, we selected one WRKY gene, BnaWRKYa to dissect molecular mechanism underlying plant-Sclerotinia sclerotiorum interaction. It wasoverexpressed ectopicly in Arabidopsis and also AtWRKYa’s expression was interfered usingRNA interference (RNAi). We have gained related transgenic lines and performed phenotypicassay of them after inoculated with Sclerotinia sclerotiorum.In addition, a systemical phenotypic assay of T-DNA insertion mutants of MAPKcascade in Arabidopsis identified a mutant showing evdient retarded root developmentcompared to wild-type. This mutant is therefore named srk1(short root kinse1). I havecloned AtSRK1gene and analyzed its function in regulating root development in Arabidopisusing a combination of techniques including molecular, biochemical and genetic approaches.