Effects Of BvM14-STPK Phosphorylation Site Mutation On Enzyme Activity Under Salt Stres

Sugar beet M14 is the monosomic additonal line by adding the No.9 chromosome of B.corolliflora Zoss.to the chromosomes of B.vulgaris L.through distant hybridization.Serine/threonine protein kinase can modulate kinase activity by autophos-phorylation or substrate phosphorylation to elicit downstream elements in response to stress.The full-length c DNA of serine/threonine protein kinase gene（BvM14-STPK）was obtained in the early study.After prokaryotic expressed,there were 31 key phosphorylation identified by LS-MC/MC,including 19 serine sites,9 threonine sites and 3 tyrosine sites.In this study,8 loci were selected from 19 serine sites for further site mutation studies.In order to avoid functional complementation of adjacent phosphorylation sites,eight key phosphorylation sites were divided into five mutant sequences（Ser31-35,Ser107,Ser216-217,Ser230 and Ser395-397）.Serine was mutated to aspartic acid（D）to mimic the phosphorylation state and phenylalanine（F）to mimic the non-phosphorylation state.In order to study the response of BvM14-STPK phosphorylation site mutated gene to salt stress,the mutated gene was ligated into p CAMBIA1300S-3×FLAG,which was used to expressed in plants,obtaining 10 recombinant vectors with FLAG-tagged and transforming into Arabidopsis STPK gene mutation strain（KO）.The 10 transgenic positive Arabidopsis with phosphorylation sites mutant compared with the Arabidopsis BvM14-STPK gene restorer（CO）,the phenotype,physiological and biochemical indexes and kinase activity were all detected under different salt concentration stress.The results were as follows:1.Ser31-35 sites of BvM14-STPK mutate into phosphorylation status(BvM14-STPK^S31DS35D)and non-phosphorylated state(BvM14-STPK^S31FS35F).Two Transgenic Arabidopsis compared with CO at 0/150 m M Na Cl treatment,the difference of the phenotype,physiological and biochemical level and enzyme activity are not significant.This indicates that the change of phosphorylation status of the BvM14-STPK protein kinase Ser31-35 site has no effect on the phenotypic,physiological and biochemical level of the transgenic Arabidopsis thaliana plants,and has no effect on the enzyme activity of the kinase.2.Ser107 sites of BvM14-STPK mutate into phosphorylation status(BvM14-STPK^S107D)and non-phosphorylated state(BvM14-STPK^S107F).Compared with CO,the phenotype and physiological and biochemical levels of the BvM14-STPK^S107Dtransgenic plants were not significantly different at 0 m M Na Cl treatment.The enzyme activity of the kinase was significantly up-regulated.Under the condition of 150 m M Na Cl,the chlorophyll content of the transgenic plants was significantly up-regulated and the leaf conductivity was significantly down-regulated.However,the phenotype and K⁺/Na⁺had no effect,and the enzyme activity of the kinase was also significantly up-regulated.Compared with CO,the phenotype and physiological and biochemical levels of the BvM14-STPK^S107Ftransgenic plants were not significantly different at 0m M Na Cl treatment.The enzyme activity of the kinase was significantly decreased.Under the condition of 150 m M Na Cl,the phenotypic differences of the transgenic plants were not significant and some physiological and biochemical levels were not observed.Both were significantly down-regulated,and the enzyme activity of the kinase was also significantly decreased.3.Ser216-217 sites of BvM14-STPK mutate into phosphorylation status(BvM14-STPK^S216DS217D)and non-phosphorylated state(BvM14-STPK^S216FS217F).Compared with CO,the phenotype,chlorophyll content and K⁺content of the BvM14-STPK^S216DS217Dtransgenic plants were significantly up-regulated under the conditions of 0 m M Na Cl and 150 m M Na Cl.The leaf conductivity and Na⁺content were significantly up-regulated,and the enzyme activity of the kinase was also significantly enhanced.Compared with CO,the phenotypic and physiological and biochemical levels of the BvM14-STPK^S216FS217Ftransgenic plants were not significantly different at 0 m M Na Cl treatment.The enzyme activity of the kinase was significantly decreased.The phenotype,chlorophyll content and K⁺content of the transgenic plants were significant down-regulation at 0 m M Na Cl treatment,leaf conductivity and Na⁺content were significantly up-regulated,and the kinase activity was significantly decreased.4.Ser230 sites of BvM14-STPK mutate into phosphorylation status(BvM14-STPK^S230D)and non-phosphorylated state(BvM14-STPK^S230F).Two Transgenic Arabidopsis plants compared with CO at 0/150 m M Na Cl treatment,phenotype and chlorophyll content and the K⁺content were significantly down-regulated while the leaf conductivity and Na⁺content were significantly up-regulated.The enzyme activity of the kinase was significantly decreased.5.Ser395-397 sites of BvM14-STPK mutate into phosphorylation status(BvM14-STPK^S395DS397D)and non-phosphorylated state(BvM14-STPK^S395FS397F).Two Transgenic Arabidopsis plants compared with CO at 0/150 m M Na Cl treatment,phenotype and chlorophyll content and the K⁺content were significantly down-regulated while the leaf conductivity and Na⁺content were significantly up-regulated.The enzyme activity of the kinase was significantly decreased.In conclusion,the phosphorylation status of Ser216-217,Ser230 and Ser395-397loci are key phosphorylation sites affecting BvM14-STPK kinase activity,which is important for studying the mechanism of BvM14-STPK protein kinase in response to salt stress.