Mining,Cloning And Functional Identification Of UDP-glycosyltransferase Genes From Rubus Suavissimus S.Lee And Angelica Keiskei(Miq.)koidz.

Many bioactive natural products of plant origin exist in the form of glycosides,which are produced by glycosyltransferases(GTs),predominantly UDP-glycosyltransferases(UGTs).Study of these enzymes has a positive effect on the understanding of the formation and utilization of the functional plant glycosides.Comparing to traditional methods of extracting natural products from plants,biosynthetic methods take advantage of high efficiency,safety and economy.With the development of biotechnology,the use of biological methods to synthesize valuable active ingredients have become an important way to improve the yield,as well as the quality of natural products.In the study of natural product biosynthesis,the use of high-throughput transcriptome sequencing provides an important platform to identify key enzyme genes,clone and efficiently express them in appropriate systems for in vitro enzymatic assay.Steviol glycosides belong to a class of tetracyclic diterpenoid glucosides which are derived from kaurene.They share a common aglycone known as steviol.Some of these ingredients are highly sweet,especially stevioside from Stevia rebaudiana extracts.Because of its high sweetness,low calorie,safety and non-toxic properties,steviol glycosides have been widely used as sugar additives in the industrial production of food and beverage,being known as the "third source of sugar in the world" following sucrose and beet sugar.Hitherto,steviol glycosides have been reported in four plant species,namely,Stevia rebaudiana and the Mexican Stevia phlebophylla A.Gray,the Chinese blackberry Rubus suavissimus S.Lee(Rosaceae),and the Japanese perennial Angelica keiskei(Miq.)Koidz.(Apiaceae).In this study,two species,R.suavissimus and A.keiskei were chosen as the research object.Based on the transcriptome data obtained from the high-throughput sequencing of these two plants,the glycosyltransferases involved in kaurane-type tetracyclic diterpene biosynthesis were studied by means of transcriptome data analysis,gene mining,cloning,heterologous protein expression and identification of enzymatic function.The main contents of this thesis include the following aspects:1)Rubus suavissimus S.Lee,also known as Chinese sweet tea or sweet leaf raspberry,belongs to the Rubus genus of Rosaceae family and is a rare natural plant in Guangxi province.R.suavissimus can produce kaurene-type tetracyclic diterpene glucoside compounds similar to that of Stevia rebaudiana,but the key UGTs involved in their biosynthetic pathways are not yet known.Based on the data of the transcripts of the leaves of R.suavissimus,191 UGT gene sequences were analyzed by bioinformatics.I selected part of these sequences(amino acids 250 to 600)with known plant UGTs to construct phylogenetic trees.Among them,62 glycosyltransferase genes with full length are screened,of which were supposed to be related to the biosynthesis of steviol glycoside compounds.Finally,40 of them were successfully cloned.By incubating with steviol glycoside substrates,six UGTs were found to show the corresponding glycosylation activity.Four of them have an enzyme activity similar to UGT74G1 that catalyze the glycosylation of the carboxyl at C-4 of the steviol skeleton.One catalyzed the 13-hydroxyl glycosylation with a similar activity to UGT85C2,and one was proposed to catalyze di-glycosylation of steviol-19-O-?-D-glucoside with an UGT91D2-like activity.The UGTs required for two-step glycosylation from steviol to rubusoside were characterized.In addition,the kinetic parameters of RsUGT41 in the conversion of steviol to steviol-19-O-?-D-glucoside were measured.2)Angelica keiskei(Miq.)Koidz.is a perennial herb belonging to the Umbellifera family.In this study,151 transcripts of putative UGT genes were systematically analyzed.Family classification and grouping of A.keiskei UGTs(AkUGTs)were performed.A total of 82 UGT sequences in the A,D,E,G,H and L groups were further analyzed and tentatively named as Ak UGT1~82.Screening of the above 82 sequences led to 23 transcripts with complete ORF and full length.After full length cloning and recombinant plasmid construction,21 sequences were obtained.90% of the cloned sequences showed > 99% matching with the original cDNA sequence.The recombinant proteins were expressed in Escherichia coli and the crude enzyme were incubated with substrate including steviol,rebaudioside A,steviolmonoside,steviolbioside and steviol-19-O-?-D-glucoside,respectively,to determine glycosyltransferase activities in A.keiskei.Finally,AkUGT49 and AkUGT50,which were similar to those of UGT74G1,were found to be able to catalyze the glycosylation of carboxyl at C-4 of steviol,producing steviol-19-O-?-D-glucoside.3)Through gene mining of transcriptome data,as well as cloning and enzymatic assay of recombinant enzymes,I finally discovered eight UGTs with activity toward kaurane-type substrate from R.suavissimus and A.keiskei.I have made a preliminary analysis of these eight sequences,such as physical and chemical properties,sequence multiple alignment and protein functional domain prediction.These works lay a foundation for further study of the functional characteristics of plant UGTs.