Cloning And Functional Identification Of Glycosyltransferase Gene In Pueraria Labota

Pueraria lobata,a traditional Chinese herb,has great medicinal value.It has been widely applied to prevent and treat cardiovascular and cerebrovascular diseases,diabetes and other disease.Isoflavonoids,such as puerarin,daidzin and genistin,are the main active compounds in P.lobata and have been proved to have antioxidant activity,anticancer activity,anti-inflammatory activity,and so on.As one of the most important tailing genes in nature,Glycosyltransferase(UGT)can transfer UDP activated sugar molecules to natural compounds and change its chemical properties.Flavonoids are significant secondary metabolites widely distributed in plant.In P.lobata,UGTs play an important role in the synthesis,transport and storage of flavonoids.Puerarin is quite special in chemical structure as it's sugar attached to the aglycone by C-C bond while most of flavinoids by C-O bond.Abviously,there are two kinds of UGT in P.lobata,one is C-glucosyltransferase(CGT)involved the synthesis of puerarin,and another one is O-glycosyltransferase(OGT)related to the synthesis of common isoflavonoids.In order to search UGT genes in P.lobata,,we conducted transcriptome sequencing.More than 140 putative UGT genes were detected,from which eight target genes were selected by homologous analysis with reference CGT and OGT protein sequences found in NCBI.This 8 UGT gene are named by GT1?GT2?GT3?GT4?GT5?GT6?GT7?GT8 in this research.Function of these genes was studied through prokaryotic expression and transgenic soybean hairy root induction.We constructed prokaryotic expression vectors and transformed them to Rosetta(DE3),then induced protein by IPTG.Target protein of GT1?GT2?GT3?GT4?GT5 ? GT6 ? GT7 ? GT8 were successfully expressed in E.coli and extracted by ultrasonication method.However,large part of protein tend to be insoluble the and appear in the precipitation.Protein activity was checked by add extracted protein to reaction assays.GT4 was found to have a strong glycosyltransferase activity and was able to catalyze the synthesis of 7-O-glucoside of daidzein,genittein,glycitein and formononetein.however,GT4 did not respond to liquiritigenin and isoliquiritigenin.Obviouely,GT4 is a 7-O-glycosyltransferase that specifically recognizes isoflavones.In addition,GT1 can catalyze the formation of formononetin,but the activity is very low.Other GT genes show no glycosyltransferase activity.We also constructed over expression vectors and transformed them to Agrobacterium rhizogenes K599.Hairy root was successfully induced by infecting soybean cotyledon with k599.Isoflavones extracted from hairy root was detected by HPLC.Compared with the control,no puerarin or other new components were found,and the content of isoflavone glucoside was not significantly different.These GTs have no function in soybean.In conclusion,CGT was not successfully synthesized in P.lobata,but a new glycosyltransferase gene was identified and it show high activity and it could be a new resource for biosynthesis.