The Metabolic Pathway And Regulation Of Flavonoids In Sweet Osmanthus Flower Petals

Osmanthus fragrans is an evergreen broad-leaved ornamental tree of family Oleaceae.O.fragrans has been cultivated since the Han Dynasty.It is widespread in the southern provinces of China.Possessing a unique aroma,O.fragrans has been widely used for culinary and medicinal purposes.O.fragrans has many varieties that are divided into four groups: Dangui,Yingui,Jingui and Siji Gui.O.fragrans produces many secondary metabolites in response to environmental stresses.These secondary metabolites play important roles in the overall metabolic activity of the plant.They also form a line of defense against pathogenic microorganisms.In this study,we used Yingui Baijie and Chenghong Dangui petals to study the flavonoid metabolic pathway of O.fragrans.The main results are as follows:We analyzed total flavonoid and quercetin contents in petals of Yingui Baijie and Chenghong Dangui via spectrophotometry and high performance liquid chromatography(HPLC).Then we compared total flavonoid and quercetin concentrations in petals of these two cultivars.Yingui Baijie was shown to have significantly higher flavonoid and quercetin concentrations than Chenghong Dangui.Using RT-PCR and fluorescence quantitative PCR,we examined expression levels of genes involved in the flavonoid metabolic pathway.Compared to Chenghong Dangui,Yingui Baijie showed significantly higher(P <0.05)expression levels of the upstream genes PAL,CHS,F3 H and FLS and much reduced expression levels of the downstream genes DFR and ANS.These gene expression differentials may be responsible to the accumulation of flavonoids in Baijie petals.We constructed 35S-Of MYB1-GFP-NOS recombinant vector,transformed Agrobacterium tumefaciens,then extracted RNA from infected O.fragrans petals.We used RT-PCR and quantitative PCR to detect the expression of genes involved in the flavonoid metabolic pathway.The over-expression of the OfMYB1 gene led to enhanced expression of PAL,CHI,FLS and decreased expression of DFR and ANS,indicating that OfMYB1 may regulate the expression of these genes.Yeast one-hybrid experiment showed that yeast co-transformed with pB42AD-ofMYB1 and pLacZi-MBP recombinant plasmids were stained blue in Z-buffer/X-gal post cultivation,while yeast co-transformed with pB42 AD and pLacZi-MBP recombinant plasmids were not.This suggested that OfMYB1 may participate in the regulation of PAL gene expression by interacting with the MYBPLANT element of the OfPAL promoter region.We constructed prokaryotic expression vector pET-30a-OfMYB1,turned it into BL21 competent cells.After cultivation and induction,protein extraction was carried out.Polyacrylamide gel electrophoresis showed that there was a 25 KD sized OfMYB1 protein.The amount of this protein increased significantly with the extension of induction time.