The Exploration Of The Functional Sites Of Phosphorylation Of BvM14-STPK Protein In Sugarbeet M14 Strain In Response To Salt Stress

M14 is the B.vulgaris monomer addition line by adding the 9^thh chromosome of B.corolliflora Zoss in the chromosome complement of B.vulgaris L.Because of the import of the 9^thchromosome of B.corolliflora Zoss makes it obtain some good genes of wild varity.It is identified that BvM14 has some excellent characters of B.corolliflora Zoss,such as drought resistance,cold resistance,salt resistance and apomixes.Also BvM14 is a good material of exploring and using of quality genes of B.vulgaris.Previously,comparative proteomics analyzise in the leaves and roots of M14 line had been performed under salt stress.And we have found that 40 differentilly expressed protein sports in leaves.Aslo we had indentied 36 differentially expressend protein spots in roots.One of them,serine-threonine protein kinase is involved in signal transduction.In this study,under the salt stress,the protein was found a 3.24 fold increase in roots.Also we thought that serine-threonine protein kinase might respond to the salt-tolerance mechanism in sugar beet M14 line.In this study,we cloned and function of serine-threonine protein kinase from sugar beet M14 line.Serine/threonine protein kinase is one of the most important classes of protein kinases,which can regulate the activity of many kinds of life through the phosphorylation of a variety of functional proteins.It was found that serine/threonine protein kinase had a significant change in salt,drought and low temperature stress,and was involved in various metabolic processes.In this study,which based on the previous work of the transcriptional group under same salt stress,a cDNA clone,named BvM14-STPK,was obtained by in PCR.The full-length oh this gene was 1 668bp,which consisted of a 1 527bp ORF.We found the gene ecoded a 508Aa protein.The protein is a hydrophilic,which belongs to the family of Protein Kinase C Family and has a ser/thr binding domain.By using the Real-time and RT-PCR technology,which found the BvM14-STPK was expressed in different M14 tissues.It’s a high level expressed in leaves.The gene could also responed to high-salt,and involved into different pathway in the roots and leaves of M14.Phosphorylation is an important reaction in the cellular life process,identification of phosphorylation sites is to determine the necessary steps of protein phospholation mechanism.In this study,we had constructed the recombinant plasmid pET28a-BvM14-STPK and put into E.coli BL21（DE3）.And western blotting had detected the fusion protein that can be produced correctly with 0.5mM IPTG and 37℃.Protein samples were digested in gel.The enriched peptodes was identified by LC-MS/MS.The MS date was obtained by searching using PD software.By comparing with green plant database matching peptide numbers,sequence coverage and quality of MS/MS spectrum,a total of 8 key Ser sites wre identified.For identification the function of Ser phosphorylation sites,segmented mutation was done.The Ser sites were mutated into Asp acid or Phe,which were sustained phosphorylation sites or dephosphorylation sites.The STPK gene function with mutated sites will be validated by transgenetic tobacco plants.