Functional Identification Of Allosides Synthase-Related Genes From Davallia Divaricata

Objective:This project aims at the enzymatic synthesis of NDP-allose active donor required by enzymatic glycosylation modification of D-allose,and to explore enzymes that can catalyze NDP-allose generation from broad enzymes reported in literature and medicinal plant,Davallia divaricate.To explore the kinases and glycosyltransferase genes involving in the NDP-allose salvage pathway from D.divaricate and complete the cloning and functional identification of genes to provide basis for biosynthesis and application of NDP-allose.Methods:In this study,RNA was extracted from D.divaricate.Based on transcrip tome sequencing technology,blast analysis and biological software Bioedit were used to screen genes with kinase and glycosyltransferase functions from the transcriptome data of D.divaricate,and further bioinformatics analysis was conducted.Then we characterized the function of the unknown genes by gene cloning,heterologous expression,enzyme purification and activity analysis.Results:Five kinases,one glucophosphomutase and two glycosyltransferase genes were synthesized from literature reports.Unfortunately,these broad enzymes did not realize the production of NDP-allose,and the experimental results showed that the substrate spectrum of these enzymes was limited.Five unknown enzymes with kinase function were screened from the transcriptome data of D.divaricate,three of which were successfully expressed and purified,one of which was identified as galactokinase,and its enzymological properties were studied.Four unknown enzymes with glycosyltransferase function were screened from the transcriptomc data of D.divaricate.Two of them were successfully expressed and purified,and identified as ADP-glucose phosphorylase and UDP-glucose pyrophosphorylase,respectively.Both of them have broad substrates,and whether allose-1-P can be catalyzed by them to form the ADP-allose and UDP-allose needs further verification.Conclusion:It is the first time to explore the kinases and glycosyltransferase genes needed in the NDP-allose salvage pathway,and we identified the galactokinase,ADP-glucose phosphorylase and UDP-glucose pyrophosphorylase from D.divaricate.The three enzymes identified in this study play important roles in different NDP-sugar active donors’biosynthsis,and the discovery of ADP-Glucose phosphorylase and UDP-Glucose pyrophosphorylase may provide direction for the synthesis mechanism of ADP-Gal,ADP-Man and UDP-Man(which has not been reported previously).In addition,ADP-glucose phosphorylase showed broad substrate specificity and has great potential to realize NDP-allose production.