Functional Identification Of OsMKK1 And OsMKK6 Genes In Rice Multiple Stress

Mitogen-Activated Protein Kinase(MAPK)cascade is a highly conservative signal transduction pathway that widely exists in eukaryotes.MAPK cascade and associated regulatory genes influence the multiple abiotic and biotic stress tolerances in plants.The growth,development,yield and quality of rice are seriously affected by adverse environmental conditions including cold,drought and salt stress.OsMKK6-OsMPK3 and OsMKK1-OsMPK4 pathway respectively is involved in the cold and salt stress tolerances,respectively.MAPK pathways have been elucidated by yeast two-hybrid assay,Bi FC and in vitro kinase assay.However,the downstream targeted protein and regulatory genes remain to be identified.In this paper,we utilized Bi FC,two-hybrid yeast assay and in vitro autophosphorylation assay to identify the MAPK pathways,by demonstrating that OsMKK1/6 spectifically interacts with MPK3/4/6,and also developing OsMKK1 and OsMKK6 overexpression and RNAi suppression expression transgenic rice lines and for their gene functions in the cold and salt stress tolerances.The main results are shown as follows.1.The subcellular localization of OsMKK1 and OsMKK6 in rice protoplast.Confocal laser scanning microscopic images indicated that MKK1-GFP and MKK6-GFP are localized into the endoplasmic reticulum,overlapping the ER marker m Cherry-HDEL,respectively.GFP,alone as a control,is distributed in the nucleus,cytoplasm,and plasma membrane.2.Bi FC assay confirmed the interactions,between OsMKK1 and OsMPK3/4,OsMKK6 and OsMPK3/4 in rice protoplast.OsMKK1-OsMPK4,OsMKK1-OsMPK6 and OsMKK6-OsMPK4 interactions were identified by yeast two-hybrid assay.3.OsMKK1 and OsMKK6 protein prokaryotic induction and kinase activity determination.The His-OsMKK1 and His-OsMKK6-SUMO fusion proteins were induced and expressed in E.coli,the in vitro autophosphorylation assay showed that OsMKK1 and OsMKK6 had autophosphorylation activity in vitro.PCR site-directed mutagenesis stategy was utilized to construct the expression vectors of autophosphorylation inactivated fusion protein genes,such as GST-MPK3-M,GST-MPK4-M and GST-MPK6-M.The results confirmed that these fusion proteins did not show autophosphorylation activity in vitro,which could be used to study their interactions with OsMKK1 and OsMKK6 in vitro.4.OsMKK1 and OsMKK6 overexpression and RNAi suppression expression transgenic rice lines were obtained by Agrobacterium tumefaciens-mediated method.T4 homozygous transgenic lines were obtained by propagation and Hygromycin selections.5.Chilling tolerance identification of OsMKK1 and OsMKK6 transgenic rice at germination stage.Under cold stress,the shoot growth of overexpression OsMKK1 and OsMKK6 transgenic rice lines improved,the overexpression OsMKK1 transgenic rice lines accumulated higher soluble sugar than the wild type.6.Overexpressing OsMKK1 significantly enhanced the chilling tolerance at seedling stage.Under cold stress,the overexpressing OsMKK1 transgenic rice exhibited the higher survival rate than wild type,the accumulation of soluble sugar was higher than that of the wild type.7.Salt tolerance identification of OsMKK6 transgenic rice at seedling stage.Under salt stress,the survival rate of overexpressing OsMKK6 transgenic rice was higher than that of the wild type.The accumulation of proline was also higher than that of the wild type.