Screening Of Interaction Proteins And Function Identification Of Protein Kinase Y82in Soybean(Glycine Max)

Plant can activate their signal transduction when adverse changes occured in the surroundingenvironment. Plant can perceive and transduce different stress signals, and activate a variety ofphysiological and biochemical systems to survive. Protein kinases play very important roles in signaltransduction, growth, development and in regulating resistant gene expression. The function of proteinkinase in signal transduction is phosphorylating the target protein. Protein interactions were required inthe process of protein phosphorylation. In this study, we isolated a protein kinase Y82from soybean andinvestigated the function of the Y82gene. We screened the interaction proteins of Y82useing the yeasttwo-hybrid system and identified the interaction between Y82and candidate proteins. This studypresents the theoretical basis for further exploring the function of Y82in stress signal transductions.1. Generation and drought identification of transgenic tobacco: The Y82gene was introduced intotobacco through agrobacterium-mediated transformation. The T1Y82transgenic tobacco lines weretreated in drought stress for3weeks. The results showed that the Y82gene improved the droughttolerance of transgenic tobacco.2. Subcellular localization assay: Y82was fused to green fluorescence protein (GFP) under thecontrol of the cauliflower mosaic virus (CaMV)35S promoter. Then the recombinant plasmids wereintroduced into onion epidermal cells. Transformed cells were incubated for18h in dark. Thelocalization of the Y82-GFP protein was observed using a confocal laser scanning microscope. Thesubcellular localization assay indicated that Y82localized into the cell membrane and nuclei,suggestings that Y82fuctions in the cell membrane and nuclei.3. Screening the cDNA library by with TPR domain: The Y82-TPR bait vector was constructed toscreen the cDNA library by co-transformation. We got six kinds of interaction proteins with Y82TPRdomain, including HSP90, protein kinases, chlorophyll A-B binding protein, ribosomal protein, abioticstress-related protein and the development-associated protein.4. Validating the interaction between Y82-TPR and HSP90s: The HSP90s in soybean was dividedinto six groups, HSP90-1, HSP90-3, HSP90-6, HSP90-8, HSP90-9and HSP90-12were cloned. Thevectors which were used to verify the interaction in yeast were constucted. The yeast two-hybridexperiment of Y82-TPR and HSP90s indicated that the TPR domain of Y82interact with HSP90-3andHSP90-9. We constructed vectors for BiFC. The transient expression assay using Arabidopsismesophyll protoplasts showed that yellow fluorescence could be observed using a confocal laserscanning microscope. This result also demonstrated that the TPR domain of Y82interact with HSP90-3and HSP90-9.