Subcellular Localization Of Four Maize Protein Kinases Responsing To Stresses In Transgenic Arabidopsis

Four maize protein kinase genes (ZmSPK1, ZmPti1, ZmASK1, ZmSIMK1) responsing to salt, mannitol, ABA or cold stresses have been cloned in our laboratory. The obtained results show that the genes may play roles in the stress signaling pathways. In order to further study their functions in vivo, the primary research on cellular compartment of the four kinases has been carried out by bioinformation and GFP (Green Fluorescence Protein) tag.With bioinformation softwares (PSORT, TargetP or MitoProt) analysis, the results show that ZmPti1 and ZmASK1 with eventual N-terminal mitochondrial targeting sequences might be localized in mitochondrial matrix, and ZmSIMK1 and ZmSPK1 might be localized in nucleus and cytoplasm.To determine the subcellular localization of the four protein kinases, using GFP as a reporter and 35S as a promoter we constructed five plant expression plasmids: pGZmX-GFP (ZmX represents the four kinases) and pG35s-GFP, and transformed them respectively into Arabidopsis by floral dip and screened stable transgenic lines by Kan resistance and PCR. Subcellular localization was determined by Confocal laser scanning microscopy. The fused protein ZmPti1-GFP is mainly localized within cytoplasm with unequal distribution, which suggests ZmPtil might localize in one special organelle. According to bioinformation softwares analysis and the size of the organelle we presumed it localized in mitochondrion. The fused ZmSPK1-GFP showed a nuclear and cytoplasmic distribution similar to that of free GFP. Compared with free GFP, ZmSPK1-GFP was more strongly accumulated in the nucleus, which suggests that ZmSPK1 might be a soluble protein and localize in the nucleus. It's a pity for failing to get comparably strong expression of ZmSIMK1-GFP and ZmSPK1-GFP and hard to achive their cellular target.The native subcellular compartment of a protein is an important characteristic with functional implications. This work will provide basis for further study on their roles in the stress signaling pathways and on their potential biological functions.