Cloning And Functional Analysis Of MYB Genes In Variegation Formation Of Tree Peony(Paeonia Rockii)

Paeonia rockii is a wild species belong to subsect.Vagintae of tree poney.Its unique and regular varigation at the base of the petal is a typical morphological feature,which have attracted extensive attention in the commercial and research fields.A large number of studies have shown that the purple variegation on the base of the peony is considered as the main source of tree peony spot,but few reports are involved in its regulatory mechanism.In this study,the variegation parts and non-spotted parts of P.rockii were used as the main experimental materials.P.ostii and three spotted peony varieties(Paeonia suffruticosa cv.‘LanHuDie’,Paeonia suffruticosa cv.‘MoChiJinHui’ and Paeonia suffruticosa cv.‘HaiHuang’)were used as control materials.In our previous study,two R2R3-MYB transcription factors MYB-1 and MYB-5 had been showed as candidate key genes for spot formation.And four structural genes related to the spot color were identified(CHS,F3’H,DFR,ANS).Differences in the process of variegation formation were determinded by quantifing the phenotype and physical and chemical properties.Real-time fluorescence quantitative analysis of the spatialtemporal expression specificity of two transcription factors in petals,combined with the trend of four structural genes and flavonoid metabolic pathway,predicted the role of MYB-1 and MYB-5 about veriagation formation.Then the gene function was veified by virus-induced gene silencing assay,which inhibited the genes expression.The results are as follows:(1)Phenotypic and physical and chemical properties analysis showed that the formation of purple spotted of P.rockii was mainly due to the accumulation of peonidin in the cell vacuoles producing red and purple colored cells.And quercetin and luteolin,as co-pigments,can deepen the purple and red in the cells.All these colored cells concentrate in the epidermis and palisade tissues at the base of the petals to form vareigation.At the same time,the base cells of the petal are ellipsoidal shape,and the content of metal elements was less,which also enhanced the base spot color.(2)View as the genetic factor,the sequences of MYB-1 and MYB-5 genes were determined by experiments.Their structure and function were predicted by bioinformatics analysis.Overexpression vectors and TRV(tobacco rattle virus)vectors were constructed to lay the foundation for functional verification.Through subcellular localization and real-time fluorescence quantitative analysis,the transcription factors MYB-1 and MYB-5 are mainlyexpressed in the nucleus.With the deepening of the spot color,the expression of MYB-1 and MYB-5 in the base of the petal showed an upward trend,which was consistent with the expression characteristics of the four structural genes(CHS、F3’H、DFR、ANS).Especially,the change of MYB-5 was more significant.When MYB-5 gene was inhibited,the expression of structural genes decreased,and the color of petal spots became lighter.Above all,In the process of verigation formation,the expression levels of MYB-1,MYB-5 and four structural genes(CHS、F3’H、DFR、ANS)increase gradually,which cause the contents accumulattion about peonidin,quercetin and luteolin.And the base of petals gradually showed purple-red spots.At the same time,cell morphology and metallic elements affect the appearance of stains.