Transcriptome-wide Mining And Functional Characterization Of Novel Flavonol Synthase Family From Ornithogalum Caudatum Jacq

The 2-oxoglutarate-dependent oxygenase is the second largest oxidase family in nature.A large group of enzymes involved in secondary metabolism of flavonoids in plants.Flavonoids have been shown to have a variety of pharmacological activities.Combined with the existing research,this study focuses on the discovery of novel and efficient flavonol synthase(FLS).A family of flavonol synthases with iron-independent flavanone 3-hydroxylase(F3H)activity was isolated from the flavonoid-rich plant Ornithogalum caudatum Jacq.1.Cloning and expression of the flavonol synthase gene of O.caudatumTranscriptome-wide mining resulted in 13 candidate genes encoding flavonol synthase.Total RNA was extracted from the bulbs of O.caudatum and cDNA was thus generated using the resulting total RNA as templates.Thirteen candidate FLS genes were then cloned by nested PCR using cDNA obtained by RNA reverse transcription as template.The prokaryotic expression vectors of 10 genes were successfully constructed and the soluble expression of 6 recombinant proteins was completed.2.Functional characterization of the flavonol synthase gene of O.caudatumAn in vitro enzymatic reaction system was constructed to verify the FLS activity of the recombinant protein with dihydrokaempferol as the substrate,and to verify the F3H activity with naringenin as the substrate.Two of the flavonol synthases had been proved to have these activities,were named OcFLS1 and OcFLS2.The OcFLSs were further explored for their broad functions with other 17 flavonoid compounds.They only showed the activities with dihydroflavones and dihydroflavonols.HPLC was used for analysis in this study,and the reaction products were further determined by LC-MS combined with the predicted standard sample.3.Enzymology characterization of OcFLSl and OcFLS2The enzymology characterization of bifunctional enzymes OcFLS1 and OcFLS2 were investigated in vitro.First,the effects of temperature and pH on enzyme activities were determined.The results showed that OcFLSs were more tolerant to low temperature than high temperature,and the optimum temperature for two functions of in vitro are all 20?.The reaction of OcFLSs were more suitable for weakly acidic environments with an optimum pH of 6.0.Then the effects of cofactors in the reaction system on the enzyme activities of OcFLSs were investigated.A novel flavonol synthase family with iron-independent F3H activity were observed.When Fe2+was not added into the reaction system,the F3H function of the two enzymes still occurred,though the activity decreased.Also,how different bivalent metal ions influence on OcFLSs were determined in vitro.Except that Mg22+ and Ca2+ had almost no inhibitory effect on the activity of F3H activity,the others had inhibitory effect on the enzyme activity,among which Cu2+ had the strongest inhibitory effect The enzymatic kinetics parameters of FLS reaction with dihydrokaempferol as the substrate and F3H reaction with naringenin as the substrate were determined for OcFLS1 and OcFLS2,and the Km and Vmax values of each reaction were obtained.4.Expression profiles of OcFLSs in various tissuesThe expression levels of OcFLS1 and OcFLS2 in roots,bulbs,leaves,flowers,bulblets and sterile bulbs of O.caudatum were detected by fluorescence real-time quantitative PCR(RT-qPCR),so as to infer the expression profiles of OcFLSl and OcFLS2 in plants.The results showed that both OcFLS1 and OcFLS2 were widely present in leaf tissues,but almost absent in bulblets,roots and sterile bulbs,which also verified the possible involvement of these enzyme in anti-ultraviolet and anti-stress functions.