Study on the Regulation of Anthocyanin Metabolism by miR 156-PhSPL3 in Pericallis hybrida
[Objective]Pericallis hybrida is an important potted ornamental flower with a variety of colors and anthocyanin metabolic branches,which is an ideal material to study the molecular mechanism of flower color regulation.miR156-PhSPL3 module is differently expressed in different varieties of P.hybrida,exploring the mechanism of this module in the formation of flower color would provide a theoretical basis for the regulation mechanism of flower anthocyanin metabolism.[Method]PhSPL3 and two Ph-miR156 precursor genes(Ph-MIR156a and Ph-MIR156b)were cloned from P.hybrida for bioinformatics analysis and tissue expression analysis.Experimental studies using VIGS,yeast one-hybrid(Y1H)and two-hybrid(Y2H)system,and dual-luciferase assays elucidated the function and regulatory role of miR156-PhSPL3 in anthocyanin metabolism.[Result]The open reading frame of PhSPL3 gene was 1 119 bp long,encoded a protein of 372 amino acids and belonged to Clade Ⅶ branch of the SPL family.Ph-MIR156a and Ph-MIR156b precursor stem-loop sequences were 108 bp and 104 bp long respectively,both containing mature miR156 sequences.Expression analysis revealed the high expression of PhSPL3 in carmine and blue varieties of P.hybrida ray florets,inversely correlated with Ph-miR156 expression.Dual-luciferase assays demonstrated that Ph-miR156 targeted to PhSPL3.Virus-induced gene silencing(VIGS)of PhSPL3 reduced anthocyanin content and downregulated expressions of anthocyanin metabolic genes PhCHS2,PhF3H1,PhANS,and regulatory gene PhbHLH17.Y2H and Y1H revealed PhSPL3 interacted with PhMYB5 and PhMYB7 and specifically bound to the promoters of PhCHS2 and PhbHLH17,thus regulating their expressions.[Conclusion]The miR 156-PhSPL3 pathway in P.hybrida may play a role in the biosynthesis of anthocyanin substances by directly regulating structural genes on the anthocyanin metabolism branch or indirectly regulating genes regulating anthocyanin metabolism.
万方数据
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