Preliminary Study On The Response Of BvM14-STPK Gene To Salt Stress

Sugar Beet M14 is the adding the No.9 chromosome monosomic addition line of B.corolliflora Zoss.to the chromosomes of B.vulgaris L.by using distant hybridization.It is identified that M14 has some excellent characteristics,such as resistance of salt,drought,cold and apomixis,which is a good material can be used to explore the quality genes of B.corolliflora Zoss.Previously,differential expression of proteins from Sugar Beet M14 response to salt stress(0,200,400m M Na Cl)were obtained by using i TRAQ LC-MS/MS.A high-throughput sequencing technique(Hiseq 2000)was used to construct the transcriptome database of sugar beet M14 in leaves and roots under salt stress(0,200,400m M Na Cl).The full-length c DNA of serine/threonine protein kinase gene(Bv M14-STPK)was obtained from the constructed RNAseq database under salt stress.Bioinformatics analysis was also be completed.Serine/threonine protein kinase can catalyze the phosphorylation of the substrate protein,also can catalyze the phosphorylation of itself,thus activating the enzyme activity of the kinase,then causing the downstream cascade reaction,participating in signal transduction process of cell.In this study,the response of Bv M14-STPK gene to salt stress was studied from the following two aspects.Firstly,we discuss the response reaction of Bv M14-STPK gene under salt stress.The plant expression vector p CAMBIA1300S-Bv M14-STPK-3x FLAG was constructed and infected into the Arabidopsis thaliana wild plants(WT)and Arabidopsis thaliana STPK mutant plants(KO),then Bv M14-STPK gene positive overexpressing plants(OX)and Bv M14-STPK gene positive recovery plants(CO)of Arabidopsis thaliana were obtained respectively.Wild type and mutant Arabidopsis as control,respectively.The phenotypic,physiological,biochemical levels and protein levels of overexpressing and recovery plants were detected under different salt stress.Simultaneously,wild type as control,transcription levels were deteted of overexpressing and recovery plants under different salt stress.The results were as follows:1.Regarding wild type plants as control,significant differences in the root length,fresh weight and dry weight of overexpressed Arabidopsis under 150m M Na Cl.Regarding mutant plants as control,significant differences in the root length,fresh weight and dry weight of overexpressed Arabidopsis under 150m M Na Cl,which indicates that the overexpression of Bv M14-STPK gene enhance the biomass of transgene plants,further proved Bv M14-STPK gene plays an important role in the growth and development of plants.2.Regarding wild type as control,significant differences in the leaf conductivity,chlorophyll content and the contents of K⁺and Na⁺.Regarding mutant plant as control,significant differences in the leaf conductivity,chlorophyll content and the contents of K⁺and Na⁺.It is illuminate that Bv M14-STPK gene plays an important role in the stability of cell membrane,the photosynthesis of plants and ionic equilibrium of K⁺and Na⁺in plant.3.Wild type as control,the expression of STPK gene in overexpression and recovery plants were detected by real-time PCR under 0m M Na Cl and 150m M Na Cl treatment,respectively.The results showed that the expression of STPK gene in overexpressed and recovery Arabidopsis changed significantly under 150m M Na Cl.This indicates that the Bv M14-STPK gene has a response to salt stress during the progress of salt-resistance.4.In order to measure the specifity of Bv M14-STPK enzyme,purified Bv M14-STPK by using FLAG tag under 0 m M Na Cl and 150m M Na Cl then measuring kinase enzyme.0m M Na Cl treatment as control,measuring the enzyme activity of overexpression and recovery plants under 150m M Na Cl.There are much more significant differences of overexpression plants enzyme activity than recovery plants.It is indicated that Bv M14-STPK kianse enzyme activity was increased,then caused the kinase cascade connection in downstream,then the resistance to salt was increased.Secondly,in order to study the influence of Bv M14-STPK phosphorylation sites under salt stress,and discuss the influence of enzyme activity was caused by variety of phosphorylation sites.p CAMBIA1300S-Bv M14-STPK-3x FLAG,the constructed recombinant expression vector was transferred into tobacco,and the expressed protein Bv M14-STPK was purified by using FLAG tag.Effect of salt stress on kinase phosphorylation site of Bv M14-STPK gene were investigated by high performance liquid chromatography tandem mass spectrometry.13 serine and 8 therione phosphorylation sites under 0m M Na Cl were identified.Also,14 serine and 7 therione phosphorylation sites under 150m M Na Cl were identified.Compared with 0m M Na Cl treatment,the results showed that the 75^th thalidine phosphorylation was disappeared and the 497^th Ser phosphorylation site was added under 150 m M Na Cl.It indicates that the phosphorylation site can be changed by salt stress.In order to investigate the influence of phosphorylation site to the activity of enzyme,we detected the activity of phosphorylation under 0m M Na Cl and 150m M Na Cl,respectively.The results show that the activity of Bv M14-STPK was increased under 150m M Na Cl.The reason of increasing Bv M14-STPK activity,caused by the disappearance of the 75^th Thr phosphorylation site,the addition of the 497^th Ser phosphorylation site,or by the interaction of them will be explored in the future.