ABSTRACT
Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6â³-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6â³RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6â³RhaT enzyme expressed in Escherichia coli exhibited 6â³-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6â³RhaT converted the administered quercetin into rutin, suggesting that FeF3G6â³RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6â³RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6â³RhaT is involved in rutin biosynthesis in common buckwheat.
Subject(s)
Fagopyrum/metabolism , Hexosyltransferases/metabolism , Rutin/biosynthesis , Chromatography, High Pressure Liquid , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fagopyrum/enzymology , Hexosyltransferases/genetics , Hexosyltransferases/isolation & purification , Phenols/metabolism , Polymerase Chain Reaction , Seedlings/enzymology , Sequence Analysis, RNA , Substrate SpecificityABSTRACT
A film-stack reaction field with a micropillar array using a motor stirrer was developed for the high sensitivity and rapid enzyme-linked immunosorbent assay (ELISA) reaction. The effects of the incubation time of a protein (30 s, 5 min, and 10 min) on the fluorescence intensity in ELISAs were investigated using a reaction field with different micropillar array dimensions (5-µm, 10-µm and 50-µm gaps between the micropillars). The difference in fluorescence intensity between the well with the reaction field of 50-µm gap for the incubation time of 30 s and the well without the reaction field with for incubation time of 10 min was 6%. The trend of the fluorescence intensity in the gap between the micro pillars in the film-stack reaction field was different between the short incubation time and the long incubation time. The theoretical analysis of the physical parameters related with the biomolecule transport indicated that the reaction efficiency defined in this study was the dominant factor determining the fluorescence intensity for the short incubation time, whereas the volumetric rate of the circulating flow through the space between films and the specific surface area were the dominant factors for the long incubation time.
