Functional Analysis Of DAM And CBF Genes During Flower Bud Dormancy In Prunus Mume

Mei(Prunus mume)shows early blooming and its ability of flowering at lower temperature is exclusively dependent on the cold response pathways and dormancy regulation of flower buds.Dormancy Associated MADS-box(DAM)genes are associated with bud dormancy and dormancy releasing,and C-Repeat Binding Factor(CBF)genes are involved in the cold response pathways.However,the molecular regulation mechanism of these genes during flower bud dormancy remains poorly defined in P.mume.Moreover,the studies of protein-protein interactions among DAMs and CBFs have not yet been reported.In this study,six PmDAMs and six PmCBFs were cloned from the flower buds of P.mume 'Sanlun Yudie'.In order to analyze the relationship of PmDAMs and PmCBFs during flower bud dormancy,quantitative real-time PCR experiments,subcellular localization tests,yeast two-hybrid assays,bimolecular fluorescence complementation(BiFC)assays,double-luciferase assays,and yeast one-hybrid assays were performed to study the expression patterns,protein-protein interactions and regulatory patterns of these genes.The main results are as follows:(1)Six PmDAMs and six PmCBFs were cloned and characterized.The expressions of these genes were detected in the annual growth processes of different organs(i.e.,flower buds,leaf buds,flowers,leaves,fruits,seeds and stems)of P.mume.The PmDAMs had two different evolutionary branches,which were PmDAM01-03 and PmDAM04-06.The PmDAMs in the same evolutionary branch showed a similar expression profile.According to the expression pattern analysis,PmDAMs and PmCBFs might play important roles in flower bud differentiation and dormancy and dormancy releasing.During dormancy and dormancy releasing of bud and stem,the similar expression patterns among these genes indicated that there might be a relationship between PmDAMs and PmCBFs.(2)The results confirmed by yeast two-hybrid assays and BiFC assays suggested that PmDAM01 and PmDAM06 could form homodimers;PmDAM01,PmDAM02,PmDAM05,and PmDAM06 could dimerize with each other;PmDAM03 could only dimerize with PmDAM06;PmCBFl could form heterodimers with PmDAM01,PmDAM02,and PmDAM06;PmCBF5 could dimerize with PmDAM01 and PmDAM06.In addition,PmCBF1 could form heterodimer with PmCBF6 and PmCBF4 could form homodimer.In P.mume,there were protein-protein interactions among PmDAMs and PmCBFs,indicating that PmDAMs and PmCBFs could not only manifest as a single protein but also form protein complexes with each other to participate in organogenesis.(3)The results of double-luciferase assays and yeast one-hybrid assays showed that PmCBFs could bind to the upstream promoter of PmDAM06.In the promoter of PmDAM06,there were four CBF binding sites.PmCBF1 could bind to four CBF binding sites(i.e.,M1,M2,M3 and M4);PmCBF3 could bind to M2 or M3;PmCBF4 could bind to M1 and M4;while PmCBF2,PmCBF5,and PmCBF6 could not bind to any CBF binding site.Based on the expression patterns,protein-protein interaction patterns and regulatory patterns of PmDAMs and PmCBFs,a molecular regulation model of these genes in flower bud dormancy was proposedIn this study,PmDAMs and PmCBFs were cloned and analyzed first,then the expression patterns,protein-protein interaction patterns and regulatory patterns of these genes were investigated,and the molecular regulation models of these genes during flower bud dormancy were suggested.These results will lay the foundation to further reveal the flowering regulation mechanism in P.mume.