| Sea buckthorn(Hippophae rhamnoides L.)is a deciduous perennial shrub of the family Elysiaceae.Seabuckthorn is rich in nutrients such as vitamins,carotenoids,lipids and flavonoids.Flavonoids are one of the most important secondary metabolites in plants,mainly in the form of flavonoid glycosides in the leaves and fruits of seabuckthorn.At present,researchs on flavonoids of seabuckthorn are mainly focuses on the identification of new aglycones and the detection of their content,while the research on glycosyltransferase and its glycosylation mechanism has not been reported.Based on the genomic information of seabuckthorn,the members of UGT gene family in sea buckthorn were identified using homology alignment and conserved domain search,and their physicochemical properties were predicted.We constructed a phylogenetic tree of sea buckthorn UGT gene family,and analyzed the motif,gene structure and replication events.The expression patterns of UGT gene in different fruit development stages of sea buckthorn were analyzed by using transcriptome data and RT-q PCR.The potential flavonoid glycotransferase gene of seabuckthorn was selected for cloning,and the prokaryotic expression system of Escherichia coli was constructed.The purified recombinant protein was used for substrate screening and enzymatic assays.The main results are as follows:A total of 89 UGT genes of sea buckthorn UGT gene family were identified,distributed on11 chromosomes and grouped into 16 phylogenetic groups.Each member of the same phylogenetic group showed similarities in the distribution of protein motif and gene structure.Tandem duplication promoted the expansion of the UGT gene family in sea buckthorn.More than half of the UGT genes were expressed during fruit development,and there were significant differences with fruit development.UGT gene may plays an important role in the formation and accumulation of fruit secondary metabolites.The specific expression of UGT gene in subspecies of sea buckthorn may be one of the reasons for the difference in the types and contents of secondary metabolites between the two fruits.In this study,p GEX-4T-1-UGT expression vectors of three sea buckthorn UGT genes La9g0469,La11g0570 and Hr UGT0002 were constructed.The best inducible conditions for the prokaryotic expression system of sea buckthorn UGT genes is 16 ℃ 22 h.With UDP-glucose as glucose donor for the GST-La11G0570 recombinant protein can catalyze the glycation of various phenolic acids and flavonoid substrates in vitro,and shows the highest activity for quercetin.The optimal catalytic condition of GST-La11G0570 was 30 ℃ p H7.5.In these conditions,the Km and Vmax of the GST-La11G0570 against quercetin were 10.12±1.32 μm and 148.34±4.88 n Kat/mg.La11g0570 was located on the F7 GT branch,which suggested that La11g0570 might be a flavonoid 7-O-glycotransferase.This work laid a foundation for revealing the biosynthesis mechanism and accumulation pattern of flavonoid glycosides in sea buckthorn.In summary,through bioinformatics analysis of the UGT gene family in seabuckthorn,three potential flavonoid glycotransferase genes La9g0469,La11g0570 and Hr UGT0002 were screened out.The catalytic activity of GST-La11g0570 recombinant protein against flavonoids and phenolic acid substrates was evaluated by prokaryotic expression system and enzyme activity assay in vitro.This study laid a foundation for revealing the biosynthesis mechanism and accumulation pattern of flavonoid glycosides in sea buckthorn. |
