Screening And Cloning Of UGTs Involved In The Glycosylation Of Flavonoids In Fortunella Crassifolia Fruits

Kumquat belongs to rutaceae Fortunella.It is an evergreen shrub or small tree native to China.Flavonoids are one of the most important secondary metabolites in kumquat fruit.Some of them have the effect of eliminating free radicals produced in the human body,preventing cardiovascular and cerebrovascular diseases,anti-aging and anti-cancer.Flavonoids are generally glycosylated in plants,which enhances the stability and water solubility of flavonoids and increases their accumulation in plants.Glycosylation modification are catalyzed by UDP-glycosyltransferase(UGT)which can catalyze transfer of sugar moiety from donor molecules to aglycone or glycoside flavonoids.Due to UGTs that usually are encoded by multiple gene family in plant genomes,it is difficult to screen those UGTs specifically involved in the glycosylation of flavonoids.In this study,we aim to identify the candidate UGTs from Liuyang kumquat fruits.We conducted qualitative and relative quantitative analysis of kumquat fruit in various developmental stages by targeted metabolomics.Then we screened highly expressed UGTs by transcriptome sequencing,and these genes further were functionally predicted by bioinformatics.The results benefit further elucidation of gene functions.The results were described bellow:1.Identification of the flavonoid glycosides in kumquat fruits by using UPLC G2-S QTOF.A total of 11 flavonoid glycosides were detected,including 2 C-glucoside flavonoids,5 C-neohesperidin flavonoids and 4 O-neohesperidin flavonoids.They are Apigenin-6,8-di-C-glucoside?3',5'-di-C-?-glucopyranosylphloretin?Luteolin-8-Cneohesperidoside?Luteolin-6-C-neohesperidoside?Apigenin-8-C-neohesperidoside?Acacetin-8-C-neohesperidoside?Acacetin-6-C-neohesperidoside?Apigenin7-Oneohesperidoside?Naringenin-7-O-neohesperidoside?Acacetin7-O-neohesperidoside?Isosakuranetin7-O-neohesperidoside.2.Alysis of relative quantification of flavonoids.The relative quantification of 5 flavnoids with higher content in the kumquat fruit were compared by liquid chromatography peak area to discover the trends of their content in the whole stage.The results showed: the content of apigenin-8-C-neohesperidoside in the peel decreased firstly,then increased and then decreased,while decreased overall in the flesh;the trend of other flavonoids were similar during the fruit developmental stages with gradual reduction and decreased dramatically during early fruit development.We also found that the content of 3',5'-di-C-?-glucopyranosylphloretin was the highest though the whole development stage.In addition,the content of flavonoids in the peels was generally higher than that in pulp.3.Transcriptome sequencing to identify UGTs with highly transcriptional levels.Based on transcriptome by using Citrus clementine as a reference,we identified 11350 genes with highly expressed levels.Among them,49 genes were thought to be FcUGTs by using annotiation and motif detection.4.Analyses of gene expression pattern.Based on the transcriptome FPKM value,we found that 49 FcUGT genes in the peels exhibited two expression patterns.The first pattern contains 24 FcUGT genes,which have a higher expression level before the “99 days after flowering”;The second is composed of 25 FcUGT genes,which showed higher expression levels after “99 days after flowering”.Compared to the peel,most of the 49 genes showed a low expression in the pulp.5.Function prediction and cloning of UGTs.The phylogenetic tree was constructed based on protein sequences of 49 FcUGT genes and 39 functionally characterized genes.We predicted FcUGTs functions according to phylogenetic clulstering and structure of flavonoid structure.The results showed that 25 highly expresed FcUGTs may be involved in the glycosylation modification of flavonoids.Finally,we cloned and sequenced eight FcUGT genes.