Screening And Identification Of Proteins Interacting With A Rice Calcium-dependent Protein Kinase OsCPK9

Rice is one of the staple food crops in the world.However,the growth of rice is constantly affected by various biotic and abiotic stress factors,which leads to the decline of rice yield and quality.During the long-term evolution,plants have formed a set of complex defense mechanisms to reduce the damage caused by various environmental adversities.Calcium-dependent protein kinases（CDPKs）are Ca²⁺binding proteins widely exist in plants.CDPKs are involved in the signaling process of plants’response to bio-and abiotic stresses.Previous studies in this laboratory found that OsCPK9 plays a positive regulatory role in response to drought and osmotic stresses as well as rice blast infection.In order to further understand the molecular mechanisms of rice OsCPK9 gene underlying plants’tolerance to biotic and abiotic stresses,the present study aims to screen and identify proteins that interact with OsCPK9.The main research and results of this study are as follows:（1）The expression vector p CAMBIA1304-OsCPK9-2×HA overexpressing OsCPK9with 2×HA tag was successfully constructed.And then,the vector was transferred into the wild type rice（Oryza sativa L.cv.Nipponbare）via Agrobacterium tumefaciens-mediated transformation method,and the the positive transgenic rice lines were obtained.（2）Total proteins were extracted from leaf tissues of positive T₁ of OsCPK9-2×HA transgenic rice seedlings.The OsCPK9 interaction proteins were screened by immunoprecipitation and mass spectrometry.The obtained proteins were analyzed by bioinformatics,and 26 candidate OsCPK9 interacting proteins were selected.（3）The yeast two-hybrid method was used to verify the interaction between the candidate proteins and OsCPK9.And the interaction between OsSDR110C-MI4 and the variable domain,kinase domain,variable domain plus kinase domain of OsCPK9 was confirmed.（4）The interaction between OsCPK9 and OsSDR110C-MI4 was verified in tobacco epidermal cells by the method of bimolecular fluorescence complementation.（5）The expression levels of OsCPK9,OsCPK19 and OsAPIP6 genes in roots,stems and leaves of rice seedlings were measured by fluorescent quantitative RT-PCR assay after treatment with ABA,PEG,H₂O₂ and Na Cl.It was found that under PEG and Na Cl treatment,the expression patterns of OsCPK19,OsAPIP6 and OsCPK9 were similar.OsCPK9,OsCPK19 and OsAPIP6 might participate in the same biological process under drought stress and salt stress.The above results provide an important reference for further research on the detailed molecular mechanism of OsCPK9’s involvement in plant stress tolerance.