Identification Of Maize ZmSTK1 Interaction Protein And Preliminary Establishment Of BiFC System

As the highest yield crop in the world,maize is an important food and feed crop.Currently,how to produce high-quality and high-yield maize is a hot issue.And the development and spread of pollen is an important factor affecting yield and quality of maize.ZmSTK1 is a cytoplasmic receptor protein kinase which specifically expressed in pollen of maize.Therefore,assumes that ZmSTK1 plays a role in pollen development and pollen tube elongation.The function of proteins is not isolated in organism which needs to work with other proteins.Therefore,exploring the interacting protein of ZmSTK1 has great significance for studying pollen development and pollen tube elongation.1.Bioinformatics analysis of the intron/exon structure of RLCK IX subfamily genes in maize,rice and Arabidopsis revealed that the majority of the RLCK IX subfamily genes have similar intron numbers.The evolutionary relationship betwween genes are closer,more similar position and phase of the introns.Accordingly,the number and phase of the gene introns in the whole subfamily are relatively conservative.2.The serine,threonine and tyrosine phosphorylation sites were predicted in ZmSTK1.The number of serine,threonine and tyrosine is 16,5 and 2,respectively.One serine and one threonine phosphorylation site are contains in the common stress domain.Only two serine phosphorylation sites in the kinase domain,but has not threonine phosphorylation site.Ser527 in the kinase domain is conserved which has a high occurrence frequency and a low mutation frequency.Therefore,predicting that Ser plays an important role in the function of ZmSTK1.3.In this study,five candidate proteins are selected.They are ENO1,HSP1,EF1-A,EF1 B and S28,respectively.Target fragments of ZmSTK1 and five candidate genes are successfully cloned.Then the ZmSTK1-pGBKT7 vectors and five candidate genes linked with pGADT7 vectors are completely constructed.4.The proteins interacting with ZmSTK1 are identified by the yeast two-hybrid method.The cytotoxicity and autoactivation assay of ZmSTK1 was performed and the results shows that ZmSTK1 has not cytotoxic to the AH109 yeast strain.The transformation of the ZmSTK1-pGBKT7 vector into the AH109 yeast strain did not activate the transcription activity of the downstream reporter gene.Subsequently,the ZmSTK1-pGBKT7 vector is respcetively co-transformed with each candidate protein vectors into the AH109 yeast strain.The result shows that there is an interaction between ZmSTK1 and ENO1.But there are no interaction between ZmSTK1 and HSP1,EF1-A,EF1 B and S28.5.The ZmSTK1-p-SPYCE(M)vector is successfully constructed,which could provide the foundation for the identification of ZmSTK1 interacting proteins by the subsequent BiFC method.