Preliminary Identification Of Pathway And Interaction Proteins Of Photoperiod Sensitive Factor ZmCCA1 In Maize

Using the maize materials of tropical and subtropical in the temperate zone.Photoperiod sensitivity is one of the major obstacles.Studying the regulation mechanism of photoperiod sensitivity key genes in the maize.It can effectively provide theoretical basis for using and improving the maize materials of tropical and subtropical.In this study,we use the Zm CCA1 of a photoperiod sensitivity gene of maize as the research object.A Yeast Two-hybrid library was constructed by using the c DNA of tropical photoperiod-sensitive maize inbred line CML288.We found the interacting proteins with the Zm CCA1 by Yeast Two-hybrid System.And we analysis the proteins' biological function.Thus providing a basis for the study of the mechanism of this gene action and look for the regulatory pathways.(1)Based on the study of Zm CCA1 in the early stage of the research group.We construct the Yeast Two-hybrid bait vector of Zm CCA1 and a Yeast Two-hybrid library by tropical photoperiod sensitive maize CML288.Screened the proteins of Yeast Two-hybrid library with Zm CCA1 bait vector.In this study,we found 43 proteins interacted with Zm CCA1 and could be found the function on the NCBI.We selected 10 proteins of relating to the light and did the Yeast rotation verification.The result of this study is that number of interaction protein is Zm00001d010811 and Zm00001d010017.(2)We found the function of the proteins that their numbers are Zm00001d010017 and Zm00001d010811.The Zm00001d010017 is a photoeriod and splicing-related protein suppressors of mec-8 and unc-52.The Zm00001d010811 is adversity response and affecting glycolysis and lipid biosynthesis-related protein SNF1-related protein kinase catalytic subunit alpha KIN10.We analysis the basic physicochemical properties,signal peptide,hydrophobicity,secondary structure and subcellular localization of SMU2.As the results,the molecular weight is 64307.33,theoretical isoelectric point is 5.82 and this protein is basic protein.This protein is stable hydrophilic and play the role in the nucleus.(3)Based on the vectors of Yeast Two-hybrid,we cloned the gene ZmCCA1 and Zm SMU2.Then,we constructed subcellular localization and Bi FC vectors.The result of subcellular localization show that two gene were localized in the nucleus.On the one hand,the Yeast Two-hybrid technique is demonstrated.On the other hand,on the physical distance this interaction is possibility;The result of Bi FC shows this two proteins have interaction.On the one hand,verifying the Yeast Two-hybrid result and on the other hand proved the result of subcellular localization.(4)Based on the materials of CML288.We found the genes' photoperiodic mode by q RT-PCR.Through analysis the results of q RT-PCR,we found CCA1 and SMU2 could have a regulatory relationship.And Zm SMU2 may further regulate the expression of CO by regulating the transcription factor VOZ.Then CO regulates the expression of FT,thereby affecting the maize's flowering.This will lay a foundation for exploring the pathway by transgenic technology.(5)In our study,we discovered a new regulatory pathway.This pathway may not be same as the original photoperiod pathway.It regulates Zm SMU2 by Zm CCA1 and then influences the function or expression of VOZ.At the last stage,Zm VOZ may regulate the Zm FT by regulating the Zm CO and ultimately affects flowering.