Identification And Functional Verification Of Flavonoid Glycoside Related Glycosyltransferase Gene In Tartary Buckwheat(Fagopyrum Tataricum)

Plant flavonoids are a class of natural compounds with various biological activities that are widely distributed in plant tissues such as flowers,fruits,and leaves.Flavonoids possess biological activities such as antioxidant,anti-inflammatory,anticancer,anti-aging,and blood pressure-lowering effects.Due to their unique chemical structure and biological activities,they have been widely applied in the fields of food and medicine.Flavonoid glycosides are compounds formed by linking flavonoids and sugar molecules(such as glucose,galactose,and rhamnose)through glycosidic bonds.They are usually present in stable forms in plants and are also one of the main forms of flavonoids in plants.Glycosyltransferases are key enzymes involved in the synthesis of flavonoid glycosides,which catalyze the combination of different flavonoid substrates and different sugar groups to form flavonoid glycosides.Tartary buckwheat(Fagopyrum tataricum)is an annual plant of the Polygonaceae family,originating from southwestern China and now widely planted in Asia,Europe,and North America.As an important medicinal and food crop of homology,Tartary buckwheat not only contains rich nutrients,but also is rich in biologically active flavonoid compounds.Currently,nearly 200 flavonoid compounds have been identified in tartary buckwheat,of which about half are flavonoid glycosides.Although some glycosyltransferase genes have been identified in tartary buckwheat and a few of them have been functionally proven to catalyze the biosynthesis of flavonoid glycosides,there are no reports on the systematic identification of glycosyltransferases involved in the biosynthesis of the six types of flavonoid glycosides in tartary buckwheat.In addition,the 3-O-glycosyltransferases and disaccharide glycosyltransferases involved in catalyzing the biosynthesis of rutin,which accounts for 70%-85% of the total flavonoid content in tartary buckwheat,have not been identified.Therefore,the systematic identification of the six classes of glycosyltransferases involved in the biosynthesis of flavonoid glycosides in tartary buckwheat,especially the identification of glycosyltransferases related to rutin biosynthesis,can not only improve our understanding of rutin biosynthesis in tartary buckwheat,but also provide theoretical basis and gene resources for future breeding of high-rutin or high-specific flavonoid glycoside tartary buckwheat.In this study,a systematic identification of the gene family of buckwheat glycosyltransferases was performed.Through phylogenetic analysis,gene co-expression,and molecular docking simulation,candidate glycosyltransferase genes involved in the biosynthesis of six major classes of flavonoid glycosides in buckwheat were determined.Based on these results,candidate genes were selected,cloned,and expressed in vitro for enzyme activity verification.The study results will further expand our understanding of the biosynthesis of flavonoid glycosides in buckwheat,providing a theoretical basis and genetic resources for future molecular breeding of high rutin or high-specificity flavonoid glycosides in buckwheat.The main research findings are as follows:1.162 tartary buckwheat glycosyltransferase genes were identified by bioinformatics methods,which were unevenly distributed across the chromosomes.A systematic evolutionary tree of the Tartary buckwheat UGT gene family and Arabidopsis UGT family members was constructed.Gene structure,promoter cis-elements,collinearity,selection pressure evolution analysis,non-biological stress response,and expression in different tissues were analyzed for FtUGTs.Results revealed that the FtUGT gene family had diverse and complex features during evolution,with many hormone-related response elements,environmental stress response elements,and MYB transcription factor binding sites.This suggests that the expression of the Tartary buckwheat UGT gene family is regulated by multiple factors such as hormones,environmental stress,and MYB transcription factors.2.A phylogenetic tree was constructed using the maximum likelihood method(ML)based on 163 FtUGT sequences and 82 known flavonoid UGTs from different plants,which identified candidate sugar transferase genes related to the biosynthesis of six major classes of flavonoid glycosides in Tartary buckwheat.Correlation analysis was performed between the transcription level of FtUGT and flavonoid glycoside metabolites to identify UGT genes related to flavonoid glycoside formation.The expression correlation between 40 flavonoid biosynthesis structural genes and126 FtUGT genes was analyzed to further identify candidate FtUGT genes related to flavonoid biosynthesis.These data provide important basic information for further study of flavonoid glycoside biosynthesis in tartary buckwheat.3.A putative tartary buckwheat UGT gene FtUGT143 that may be involved in the biosynthesis of flavonoid C-glycosides was successfully cloned using RT-PCR technology.The encoded protein sequence of FtUGT143 consists of 224 amino acid residues and is a stable hydrophobic protein.The codon preference pattern of FtUGT143 was analyzed,and the homologous modeling and molecular docking simulations of FtUGT143 protein were performed.The results showed that FtUGT143 can interact with the substrate,vitexin,and isovitexin to synthesize the C-glycosylated flavonoids,orientin,and isoorientin,indicating that FtUGT143 may be involved in the biosynthesis of flavonoid C-glycosides in tartary buckwheat.Fluorescence quantitative expression analysis showed that FtUGT143 is mainly expressed in the leaves,stems,and roots of tartary buckwheat during the seedling stage,providing a theoretical basis for the subsequent protein functional validation and directional mutation of FtUGT143.4.A key UGT gene FtUGT163 involved in the biosynthesis of rutin in tartary buckwheat was successfully cloned using RT-PCR technology.FtUGT163 was analyzed,and a prokaryotic expression vector was constructed.The prokaryotic expression protein was expressed and purified,and in vitro enzyme activity experiments were performed.The results showed that FtUGT163 has UGT activity and can catalyze the formation of quercetin-3-O-rutinoside from quercetin.This reveals the catalytic mechanism of FtUGT163 and improves the pathway of rutin biosynthesis.