Study On The Autophosphorylation And Universal Substrate Phosphorylation Of Sugar Beet BvM14-STPK Protein Kinase Under Salt Stres

Protein phosphorylation is an important type of post-translational modification.Protein kinases catalyze autophosphorylation and phosphorylate substrate proteins to activate or inhibit substrate protein activity,allowing plants to respond to a variety of biotic and abiotic stresses.The research team used i TRAQ LC-MS/MS technology to obtain the differentially expressed protein of the sugar beet M14 line,and obtained a protein that was up-regulated under salt stress,namely serine/threonine protein kinase（Serine/Threonine protein kinase,BvM14-STPK）.By purifying BvM14-STPK from prokaryotic and plant expression systems,31 phosphorylation sites were identified by mass spectrometry and 8 key serine phosphorylation sites were selected,the 230th Ser is close to the DFG of the BvM14-STPK kinase activation loop（Asp231-Phe232-Gly233）motif,DFG catalyzes the transfer of phosphate groups to enable autophosphorylation and phosphorylation of substrates,In this study,Ser230 was mutated to aspartic acid（D）and alanine（A）,to mimic respectively the phosphorylated and non-phosphorylated states of Ser230.In this study,BvM14-STPK was constructed as a prokaryotic expression vector with His tag and transformed into E.coli BL21.The target protein was expressed in soluble form after induction by at 12℃and 0.5 m M IPTG.Ni²⁺resin affinity chromatography was used to purify the soluble protein.The results of phosphorylation experiments in vitro with ³²P isotope labeling showed that BvM14-STPK in prokaryotic cells had self-phosphorylation and strong phosphorylation activity to the general substrate myelin basic protein（MBP）.BvM14-STPK was used to construct plant expression vector with FLAG tag.Flower-staining method was used to transform Arabidopsis thaliana and obtain transgenic positive seedlings,Western blot was positive.The target protein was purified by magnetic beads of FLAG tagged antibody.In vitro phosphorylation results showed that the plant expression system BvM14-STPK did not detect its autophosphorylation activity,the phosphorylation activity of BvM14-STPK to MBP substrate increased after salt stress.Secondly,BvM14-STPK^S230Dand BvM14-STPK^S230Amutant genes at Ser230 locus were used to construct prokaryotic expression vectors.Compared with the BvM14-STPK,BvM14-STPK^S230Dhad no autophosphorylation activity and could not phosphorylate MBP,that is BvM14-STPK^S230Dcompletely lost its kinase activity.BvM14-STPK^S230Ahas weak autophosphorylation activity and MBP phosphorylation activity in prokaryotic cells.BvM14-STPK^S230Dand BvM14-STPK^S230Amutant genes of Ser230 locus were used to construct plant expression vectors.Compared with BvM14-STPK,BvM14-STPK^S230Dand BvM14-STPK^S230Ahad no autophosphorylation activity,BvM14-STPK^S230Dhad no substrate phosphorylation activity,and BvM14-STPK^S230Ahad weak substrate phosphorylation activity to MBP after salt stress.The above results showed that BvM14-STPK had kinase autophosphorylation and MBP phosphorylation activity in prokaryotic expression system,and the relationship between kinase autophosphorylation activity and substrate phosphorylation activity was interactive.Kinase autophosphorylation activity was not detected in plant expression system,and salt stress could induce the activity of BvM14-STPK kinase.Natural state kinase phosphorylated by 230-site serine has the highest activity and can not be replaced by other amino acids.This study lays a foundation for further research on BvM14-STPK kinase interacting protein and provides important theoretical and experimental guidance for the study of salt resistance mechanism of BvM14-STPK gene.