Functional Analysis Of Stress-responsive And Molecular Biological Analysis Of Protein Kinase GmPK05-1 In Soybean

High temperature,drought,and high salinity are common stress conditions that adversely affect crop growth and development.These stresses cause metabolic abnormality and lack of nutrition element in plant cells.Using genetic engineering and molecular biology methods to mine stress-induceble gene and obtain the stress-tolerant crops in one of the most important aspects in modern agriculture.Protein kinase is an important element of plant signal transduction systems.Protein kinase may be reversible through the phosphorylation to participate in stress signal transduction,thereby further respond to external stresses on plants.To further understand Protein kinase stress-resistant molecular mechanism and utilize stress-resistant genes,soybean protein kinase gene GmPK05-1 were isolated based on a cDNA library of seedlings.We intensively study the functions of target genes used transgenic arabidopsis GmPK05-1 material and mutants of the gene in arabidopsis in abiotic stress response and study the kinase in arabidopsis subcellular level of expression and the modification of the protein levels.Using the GmPK05-1 protein as bait,we screened for GmPK05-1-interacting proteins by yeast-2-hybrid system.Therefore,further investigating the interactions among GmPK05-1-interacting proteins may help resolve signal transduction systems.Some defense-relate genes were identified with cDNA library and they enriched the stress-induceble gene resources.Our results are mainly as follows:1)The GmPK05-1 protein kinase gene in arabidopsis thaliana,excessive expression of strain and the mutant plant adversity resistance phenotype analysis.We preliminary judgment this protein kinase involved in the response salt,drought,and such abiotic stress may in these signaling plays an important role.2)Mediated by PEG method to convert GmPK05-1-GPF to arabidopsis protoplast,observe its positioning in arabidopsis central cells.Results showed that the protein GmPK05-1 length positioning on the cell membrane.3)Through the building to the prokaryotic expression vector pColdTM-TF-GmPK05-1,IPTG induction express and purify fusion protein his-GmPK05-1,using three kinds of antibody to phosphorylation Western experimental verification since kinase phosphorylation activity,the experimental results show that GmPK05-1 in tyrosine phosphorylation site can occur itself.4)By yeast two hybrid methods,the bait plasmid pGBKT7::soybean drought and salt GmPK05-1 library plasmid pGADT7::cDNA into yeast AH 109,comparative analysis after a preliminary screening of the soy protein kinase GmPK05-1 the interactions of candidate proteins,on the basis of prediction may pick out four physiological function of protein interactions,will these four protein respectively named GmMYB54,GmCK2,GmF07,GmCA14.5)By double fluorescent complementary technology(BiFC)and pull down further validation of protein interactions,the results show that in vivo and in vitro GmPK05-1 and GmMYB54 can interact with each other,by yeast two hybrid and BiFC validation protein interactions,the results show that the GmPK05-1 and GmCK2 protein interactions.6)Mediated by PEG methods will GmMYB54 GPF and GmCK2 GPF into arabidopsis protoplast,observe the subcellular localization,results show that the GmMYB54-GPF and GmCK2-GPF positioning on the cell membrane and the nucleus.