Cloning Of Flavonoid 3'-hydroxylase Gene From Dracaena Cambodiana And Funcitional Identification Of Its Promoter

Dragon's blood is a rare traditional medicine mainly derived from Dracaena cambodiana and Dracaena cochinchinensis.It is red resin produced by the plant after an exogenous stimulation and injury.The chemical constituents and pharmacological activities of dragon's blood have been extensively studied,but research on its biosynthetic mechanism remains unknown.In this study,we cloned a DcF3'H and its promoter,which is one of the key genes on flavonoid biosynthetic pathway,aim to understanding the biosynthesis of flavonoids in.cambodiana.DcF3'H(Flavonoid 3'-hydroxylase)which have a full-length of 1877bp,contains a 287bp intron and a 1533bp open reading frame that encodes 510 amino acids.DcF3'H contained p450 conserved domains.Protein sequence alignment and phylogenetic analysis showed high identities to the F3'H from Narcissus tazetta and low identities from Medicago truncatula and Cicer arietinum.F3'H expressed highest transcription in the flowers and lowest in the roots.DcF3'H transcription was up-reg?lated by 6-BA but down-reg?lated by MeJA.The promoter region of DcF3'H was isolated,which contains many hormonal response element,a variety of light-responsive element,and the MYB binding sites MBS.We use Dual luciferase reporter system for detecting the activities of different DcF3'H promoter fragments,and F3'HP2 showed highest activity in all segments of the promoter.6-BA enhanced the activity of DcF3'H promoter significantly,especially the promoter region-756bp to-465bp;but MeJA have exactly the opposite effect.There are interactions between DcF3'H and DcbHLH by yeast one-hybrid technology.Then we use tobacco protoplasts as a plant expression system,further identified the interactions between the DcF3'H promoter and DcbHLH,and DcbHLH can enhance the activity of DcF3'H promoter.