The Expression,purification And In Vitro Kinase Activity Assay Of A Calcium-dependent Protein Kinase,OsCPK9,from Rice

Rice（Oryza sativa L.）is an important food crop in the world.Various adverse environmental stresses such as drought,salinaty and extreme temperatures could cause significant reduction in rice production,threatening food safety.Studies have shown that plant calcium-dependent protein kinases（CDPKs）play important roles in plant response to various stresses.Previous research work in this laboratory showed that OsCPK9,a member of the rice CDPKs family,could respond to drought,osmosis,and dehydration stresses,and OsCPK9-overexpressing transgenic rice are tolerant to drought stress.CDPKs,including OsCPK9,exert their physiological functions depending on its kinase activity.The present study aims to express OsCPK9 and to explore its kinase activity through in vitro experiments.The research content and results are as follows:（1）Three prokaryotic expression vectors p ET28a-OsCPK9（full-length gene）,p ET32a-OsCPK9-KD（kinase domain fragment）,and p ET28a-MBP-OsCPK9（full-length gene fused with MBP tag）were successfully constructed.（2）Among them,the expression vectors p ET28a-OsCPK9 and p ET32a-OsCPK9-KD were transferred into E.Coli Rosetta-gami（DE3）p Ly S expression strain,while p ET28a-MBP-OsCPK9 was transferred into BL21（DE3）expression strain.After induction by IPTG,all the target proteins,OsCPK9,OsCPK9-KD and MBP-OsCPK9 can be expressed correctly.（3）We found that OsCPK9 and OsCPK9-KD existed in the insoluble inclusion body,and MBP-OsCPK9 partially existed in the bacterial lysis supernatant in a soluble form.The above proteins were purified by Ni²⁺-NTA affinity chromatography column,and finally the three kinds of target proteins with high purity were obtained.（4）The results of in vitro kinase activity experiments showed that OsCPK9 and OsCPK9-KD had autophosphorylation activity.After fusion with MBP tag,soluble MBP-OsCPK9 had Ca²⁺-dependent autophosphorylation activity and could catalyze the phosphorylation of the general kinase substrates MBP and HistoneⅢ,indicating that OsCPK9 is an active Ca²⁺-dependent protein kinase from rice.Results of present study laid a foundation for screening and identification of OsCPK9kinase substrates,and for further exploration of in vivo activation mechanism and biological function of OsCPK9.