Functional Analysis of SoF3'H in the Synthesis of Anthocyanidin in Syringa oblata Petals
Flavonoid-3'-hydroxylase(F3'H),a key enzyme in the anthocyanidin synthesis pathway,plays an important role in plant flower coloration.In this study,in order to further explore the role of F3'H in flower pigment synthesis in lilac(Syringa oblata),the SoF3'H was cloned,and the protein homology sequence comparison and phylogenetic tree were examined.qRT-PCR in different flower developmental stages and organs was used to analyze the SoF3'H expression patterns.Functional validation was performed by transient silencing and overexpression of the SoF3'H gene in the lilac petals.The promoter of SoF3'H was cloned and analyzed,the yeast one-hybrid assay was performed to verify the binding of SoAN2 and SoF3'H promoter,and the expression patterns of SoAN2 was performed.The results showed that the coding sequence of SoF3'H was 1 587 bp,putatively encoding for 528 amino acids.SoF3'H has a conserved domain of p450 superfamily,belonging to the cytochrome P450 family,and has the highest similarity(82.46％)with Olea europaea.Accompanied with flower development,the petals were discolored and the expression levels of SoF3'H was decreased gradually.In different organs at flower full blossom stage,the highest expression level of SoF3'H was found in corolla lobes,and relatively lower expression occurred in roots,stamens and stems.The silencing of SoF3'H resulted in significant fading of petals and remarkable reduction of anthocyanidin content.Contrarily the overexpression of SoF3'H led to significant coloration of petals and obvious increased of anthocyanidin content.Then,the 2 000 bp long promoter sequence of SoF3'H was cloned,which has MYB binding site.SoAN2,a member of MYB family,was confirmed to bind to this promoter directly and the expression of SoAN2 at different flower development stages tended to decrease.The results further suggested that SoAN2 can promote the expression level of SoF3'H to increase anthocyanidin content regulating petal coloration.
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