Cloning And Functional Identification Of Flavonoid-C-Glycosyltransferase Genes In Phyllostachys Edulis

Phyllostachys edulis is the most widely grown and most economically valuable bamboo species in China.The leaves of P.edulis are rich in flavones such as valerin and vitexin.However,there is no specific study on the glycosyltransferases that synthesize these four flavonoids.In this study,the glycosyltransferase fusion protein of P.edulis was successfully obtained by prokaryotic expression system and eukaryotic expression system.And then the function of the protein was identified by an enzyme-catalyzed reaction.This study further recognized the formation of flavonoid glycosides by flavonoid-C-glycosyltransferase in P.edulis,and it provided a reference for the biosynthesis technology of flavonoids in the future.The main results of this study were as follows:1.Based on the sequencing of the bamboo gene library and the bioinformatics analysis technology,three flavonoid-C-glycosyltransferase genes were obtained from the bamboo gene pool,and named Pe CGT1,Pe CGT2 and Pe CGT3.Through the alignment analysis,they belong to the glycosyltransferase GT1 family.According to phylogenetic tree analysis,Pe CGT1 and Pe CGT2 have high similarity,which has high homology with CGT of Brachypodium distachyum and Aegilops tauschii,while Pe CGT3 has the highest homology with CGT of Indosasa hispida.Through physical and chemical properties and structural analysis,they are all stable hydrop Hobins and their spatial structure is GT-B.2.A total of five prokaryotic expression vectors were constructed in this study,namely p ET24a-Pe CGT1,p ET24a-Pe CGT2,p ET32a-Pe CGT1,p ET32a-Pe CGT2 and p ET22b-Pe CGT3.They were transferred into E.coli BL21(DE3)and Rosetta(DE3).p ET24a-Pe CGT1 and p ET24a-Pe CGT2 were not expressed in both E.coli expression systems.p ET32a-Pe CGT1,p ET32a-Pe CGT2 and p ET22b-Pe CGT3 were expressed in both systems.3.Since the proteins of interest expressed in E.coli were inactive,two eukaryotic expression systems were constructed in this study,namely p PIC9K-Pe CGT1 and p PIC9K-Pe CGT2.Between the two,Pe CGT2 was not successfully expressed,and Pe CGT1 obtained an active protein of interest.The target protein was produced after 48 hours,and he reached the highest protein content after 96 hours.Then successfully obtained purer Pe CGT1 protein by protein purification and ultrafiltration concentration technology.It was known by enzyme-catalyzed reaction that Pe CGT1 catalyzes the formation of two products with2-hydroxynaringenin as a substrate.Glycosyltransferase plays a crucial role in the growth and development of plants,but the properties of glycosyltransferase genes and their proteins in bamboo have not been studied in detail yet.This study successfully obtained a glycosyltransferase from P.edulis and we have strengthened its activity,which laid a foundation for further research on glycosyltransferase in bamboo.