Functional Verification Of The Buckwheat Flavonoid Glycosyltransferase Gene FtUFGT

Tartary buckwheat(Fagopyrum tataricum),the scientific name of Tartary buckwheat,a dicotyledonous plant of the Polygonaceae family,is an important traditional minor cereal and medicinal food plant in China.Tartary buckwheat flavonoids are important natural sources of plants,flavonoids are considered powerful antioxidants in plants,followed by flavonoids that have a better medicinal and economic value by the increasing attention and love of people.Glycosylation reactions are key modification steps occurring during the synthesis of plant natural products and are key reactions that determine the chemical complexity and diversity of plant natural products and affect their chemical properties and biological activities.Also,glycosyltransferases are involved in the regulation of plant growth and development in response to abiotic stresses.In order to deeply analyze the role of glycosyltransferases in the functional mechanism of the Tartary buckwheat flavonoid synthesis pathway,this study was conducted to functionally validate the Tartary buckwheat flavonoid glycosyltransferase FtUFGT3 gene.The results obtained from this study are as follows:(1)The pET28a-FtUFGT3 vector was constructed,and the recombinant protein FtUFGT3 was induced at 22℃,0.1 mM IPTG,and the induction time was 8 h.The induced recombinant protein was also detected by enzymatic assay in vitro,and the results showed that the recombinant protein FtUFGT3 could use kaempferol as the substrate and UDP-glucose as the sugar donor to form kaempferol-3-O glucoside(astragalin)and cannot catalyze kaempferol to form kaempferol-3-O-galactoside with UDP-galactose as the sugar donor,in addition,FtUFGT3 cannot catalyze dihydrokaempferol and dihydroquercetin with UDP-glucose and UDP-galactose as the sugar donors.This result suggests that the FtUFGT3 gene shows broad substrate specificity but can only catalyze the formation of the corresponding 3-O-glucoside with UDP glucoside as the sugar donor.(2)The overexpression pCAMBIA1306-FtUFGT3 vector was constructed to genetically transform Arabidopsis thaliana,and the total flavonoid and flavonoid content was analyzed in the overexpression Arabidopsis.The expression of key genes in the Arabidopsis flavonoid pathway was detected.The results showed that the total flavonoid content in the overexpressed pCAMBIA1306-FtUFGT3 strain was significantly higher than that of the wild type,and had a significant effect on the expression of key genes in the flavonoid synthesis pathway,significantly increasing the expression of the upstream gene At PAL and the downstream gene At FLS,which was hypothesized to be the main factor for the overexpression of FtUFGT3 gene to increase the total flavonoid content in Arabidopsis.(3)Arabidopsis thaliana lines overexpressing pCAMBIA1306-FtUFGT3 were subjected to cold stress treatment at 4℃ for 96 h.The total flavonoid content and flavonoids of Arabidopsis thaliana were analyzed under cold stress treatment,as well as the detection of antioxidant capacity and stress gene expression levels in the overexpressed Arabidopsis thaliana strain.The results showed that the total flavonoid content was significantly increased in the overexpression strain under cold stress,and the antioxidant capacity of the overexpression strain was higher than that of the wild type by DPPH and ABTS radical scavenging ability.The detection of stress gene expression levels showed that overexpression of the FtUFGT3 gene significantly increased the expression of stress-responsive genes in Arabidopsis.