Function Identification Of A Gene Encoding UDP-7-O-Glucosyltransferase In Tea (Camellia Sinensis(L.)O.Kuntze)

In tea plant,the flavonoids compounds are the most important secondary metabolites.In addition,the glycosylation flavonoids are the main stable conditons.Many UDPG-glycosyltransferase(UGTs)that are involved the glycosylation derivative reaction of flavonoids and phenolic acid compound.In previous works,we had isolated a candidate gene that may play flavonoids glycosidic synthesis role in tea plants.The transcription level of this gene could be induced by exogenous agent including SA、MeJA and mannitol,which indicated that the function of this gene is involved in response to defensive and stress reaction.The gene was cloned and submitted to NCBI(accession KP682364),which named as CsUGT75L12 by the UGT nomenclature ID.The maltose binding protein(MBP)fusion protein of CsUGT75L12 were expressed in E.coli Novablue(DE3),and the recombinant protein was purified by affinity chromatography on a amylose resin.The purified rCsUGT75L12 protein was prepared for the enzymatic activity assay and suggested that the glucosylation took place in hydroxyl group at the C7 position of flavonoids substrate;Meanwhile,the quercetin 3-O-α-rhamnoside-7-O-β-glucoside and kaempferol 3-O-α-rhamnoside-7-O-β-glucoside belong to 7-O-glycosidic by detected in transgenic Arabidopsis overexpressed CsUGT75L12.In this study,we found that some amino acid residues and certain regions were important to enzyme activity of CsUGT75L12 by a series of site-directed mutagenesis and truncation experiments.The study supplied a new horizon that improve our perception for the mechanism of the defensive and stress reaction in tea plants,which is important for the selective breeding of good varieties and regulation of quality.The main results are as follows:1.The ORF sequence of CsUGT75L12 gene were obtained by the transcriptome data analysis of tea plant and 3’/5’-RACE were cloned.The molecular weight of the protien is 56.74 kD.The alignment of the amino acid sequence of CsUGT75L12 were obtained with VvGT1 and above-mentioned homologues,which displayed conservation in UDP-binding domain of the PSPG box.Most noticeable of all,there was a longer N-terminal(39 aa)in the amino acid sequence of CsUGT75L12 compared to other genes.It is shown that CsUGT75L12 groupe to cluster Ⅱ with some anthocyanin 5-O-glycosyltransferase from the phylogenic tree.2.The transcriptional expression of CsUGT75L12 surveyed in different organs and different inductive agent treatment using qRT-PCR analysis.The result showed that CsUGT75L12 was expressed in most tissues and organs of tea plant,particularly expressend in shoot and the third leaves,but not in the root.Exogenous induced expression of CsUGT75L12 was analysed in a time course.The result of exogenous treatments showed that the transcript level of CsUGT75L12 was significantly up-regulated under the treatment of methyl jasmonate(MeJA),SA and mannitol.3.Recombinant plasmid of PMAL-CsUGT75L12 was correctly constructed,and the maltose binding protein(MBP)fusion protein of CsUGT75L12 were expressed in E.coli Novablue(DE3).The recombinant protein was obtained through the induce of IPTG,and purified by affinity chromatography.The result of enzymatic activity showed that the rCsUGT75L12 can specially recognize the flavonoid(naringenin,apigenin,kaempferol,and genistein)as the sugar acceptors,but no products were detected when applied the anthocyanin and catechin.The result of enzymatic activity shown that the UDP-Glc as the main sugar donor.The result according to H nuclear magnetic resonance suggested that the glucosylation toke place in hydroxyl group at the C7 position of kaempferol substrate.4.The open reading frame of CsUGT75L12 was subsequently shuttled into the pCB2004 vector to construct the PCB2004-CsUGT75L12 recombinant plasmid.The correct construct was transformed into the wild type(WT)of Arabidopsis thaliana ecotype Columbia-0(Col-0),and the F2 homozygote seeds was obtained through screening and identifying.The UPLC-QQQ-MS/MS analysis shown that the components of quercetin 3-O-α-rhamnoside-7-O-β-glucoside and kaempferol 3-O-α-rhamnoside-7-O-β-glucoside can accumulate in seed of transgenic Arabidopsis overexpressed CsUGT75L12 seeds,and the expression of gene is coordinate with the accumulation fo its components.5.Homology models of CsUGT77L12 was constructed with VvGT1(2c9z)as the crystal template model.In order to search the significance of some key amino acid residues and certain regions for enzyme activity,the model of CsUGT75L12 were built with the molecule of kaempferol and UDPG,respectively.When truncation out the N-terminal 39-peptide of the CsUGT75L12,the activity for UDP-Gal was observably increased,but UDP-Glc was not changed.The result suggested that the N-terminal regions may be related to the specificity of sugar donors.Many studies reported that the amino acid residues of H56 and T151 are necessary for UGTs in plants.The result of crystal homology models showed that the two residues localed at the key position of the substrate pocket.But the experimental of point mutation experimental result showed that the enzyme activity observably increased when the single mutation happened in H56A and T151A.Meanwhile,some key amino acid residues identified by point mutation experimental,can participate in the binding of sugar donors and receptors with CsUGT75L12.