The Terpenes Glycosyltransferase Function Analysis And Diurnal Rhythm Of Glycoside In Osmanthus Fragrans Lour.

Osmanthus fragrans Lour.Is a traditional Chinese flower and an important garden plant.It has significant market value on landscape and processing industry due to its unique aroma.Terpenoids,which are the key characteristic aroma substances,could forms emission,acclumulation and glycosylated volatiles in 0.fragrans plant.Emission volatile can be directly released and perceived.Glycosylated volatiles are considered to be storage of aroma substances,composed of aglycone and glycone which are storage forms of terpenoids can be formed and released by the action of endogenous enzymes such as dehydrogenases and glycosyltransferases.Previous studies have shown that the terpene substances in O.fragrans are converted into odorless glycosides and are the most valuable aroma precursors in the processing and production.Recently,there are few reports of glycosylated volatiles and related enzymes in O.fragrans.Therefore,this study first isolated and identified of the glycoside aroma from O.fragrans ‘Liuye petals’,and combined the volatile and free aroma substances to summarize the diurnal rhythm of glycoside volatiles.In addition,the preliminary functional verification and expression analysis of terpene glycosyltransferase gene were studied on the basis of the transcriptome of O.fragrans,and the formation rule of the terpene glycoside volatiles in t O.fragrans was summarized,which provided a theoretical basis for the utilization of O.fragrans.1.The analysis of Aglycone and Glycone of Glycoside Volatiles in O.fragrans ‘Liuye’O.fragrans ‘Liuye’ was used as material to analyze the aglycone and glycone of glycoside volatiles.In O.fragrans ‘Liuye’ flowers 20 aglycone conpounds were detected in glycoside volatiles,grouped into monoterpene alcohol,monoterpene ketone,monoterpene oxide and aromatic compounds.Glycoside volatiles were abundant with 4-hydroxy-β-Ionone,3-Oxo-α-ionol and benzeneethanol,4-hydroxy-.Glycosides are mainly monoterpenes,and 9 glycosylated monoterpenes are also detected in the volatile.The aglycone of glycoside volatiles mainly are glucose,xylose and rhamnose.2.Screening of Glycosyltransferase Gene in Osmanthus fragrans TranscriptomeOn the basis of transcriptome of O.fragrans ‘Liuye’and O.fragrans ‘Gecheng’,we Screened the glycosyltransferase gene of terpenoid volatiles.We screen 687 and 296 unigenes which were annotated glycosyltransferase in two Osmanthus fragrans transcriptome.And then according to the PSPG box in the plant’s glycosyltransferase family 1 associated with secondary metabolism and the complete domain of glycosyltransferase,we screened 182 and 53 unigenes.According to phylogenetic tree analysis we screened out belonging to the glycosyltransferase 85 family unigenes.We construct phylogenetic trees with unigenes of belonging to 85 family and the reported terpene glycosyltransferase gene.And then five unigene sequences were screened for follow-up experiments according to the sequence homology of the terpene glycosyltransferase gene that has been reported.3.Functinoal Analysis and Subcellular Localization of OfUGT1-4In this study,based on the sequences of five glycosyltransferase genes in transcriptome,the full length of four gene sequences were cloned from the c DNA of Osmanthus fragrans flower by RACE-PCR.,and named as OfUGT1,OfUGT2,OfUGT3 and OfUGT4 respectively.The phylogenetic tree analysis of these four glycosyltransferases showed that they all belonged to the 85 family.Through the transient expression of the tobacco,we found that the four genes were simultaneously located in the cell membrane and nucleus.OfUGT3 and OfUGT4 are proved that could glycosylated linalool oxides by infiltrated with over-expression vector and the volatile aglycones into tobacco leaves.4.The Circadian Rhythm of Glycoside Volatiles and Transcriptional expression of related gene in ‘Liuye jingui’The changes of compounds of glucoside volatiles in ‘Liuye jingui’were different on three days different times.But the compounds in glycoside volatiles had a significant circadian rhythm,glycoside gradually increased from 6:00 and got the top at 18:00 or00:00.The gene levels of OfUGT1-4 and 13 genes involved in the monoterpene synthesis MEP pathway were analyzed at four different time points in three consecutive days.The results showed that the expression of OfUGT1,OfUGT2 and OfUGT3 gene had obvious circadian rhythm.The gene expression was corresponded with the contents of aromatic compounds suggested that the genes in MEP pathway play the important role on fomation of terpene compounds.The different expression level of gene at different flowering stage showed the gene expression of OfUGT1,OfUGT2 and OfUGT3 was the highest in the bud period,and gradually decreased with the flowering period.There was no significant change in the expression of OfUGT4 at each flowering stage and daytime and night.