Screening For Interaction Protein With Arabidopsis AtSnRK2.4 By Yeast Two-hybrid System

Sucrose non-fermenting-1(SNF1)-related protein kinase 2(SnRK2)are serine/threonine-specific protein kinase which exists only in plant.SnRK2 plays major roles in signaling transduction and in response to environmental stress such as salt and drought,as well as some essential physiology processes.AtSnRK2.4,a SnRK2 protein kinase family member in Arabidopsis thaliana,was used as the research object in this study.We used AtSnRK2.4 as bait,using a yeast two-hybrid system to screen for the interacting protein,identifying interaction between two proteins,and to lay a foundation for exploring the resilience mechanism.In this study,we cloned AtSnRK2.4 gene into the pGBKT7 vector,and named recombinant plasmid as pGBKT7-AtSnRK2.4.Here,2-10 was finally identified as a interacting partner of AtSnRK2.4 through a yeast two-hybrid assay.The interaction between 2-10 and AtSnRK2.4 was confirmed by a pull-down assay in vitro and a bimolecular fluorescence complementation assay in vivo.GST-AtSnRK2.4 can phosphorylate itself.Using GFP fluorescence labeling technology,the fluorescence signals of AtSnRK2.4-GFP and 2-10-GFP were similar to the GFP signals and were presented in cytosol into onion(Allium cepa)epidermal cells.Finally,real-time quantitative PCR was conducted to analysis of the expression of AtSnRK2.4 and 2-10 gene at mRNA level.Expression of AtSnRK2.4 and 2-10 gene were induced by salt,ABA and mannitol,and changed with the time extending,which suggested that there were some response relationship between the expression of AtSnRK2.4 and 2-10gene and environment(Salt,ABA and drought)stress.It laid a foundation for further studying molecular mechanism of AtSnRK2.4 in response to adverse environment such as salt and drought.