Abstract: [Objective] C-glycosyl flavonoids are abundant in Phyllostachys edulis leaves and possess vital biological activities. C-glycosyltransferase (CGT) is the key C-glycosylation enzyme in C-glycosyl flavonoid biosynthesis. However, no reports currently exist on the C-glycosylation pathway and C-glycosyl flavonoid CGT from P. edulis leaves. In this work, The main aims of this work were to analyse and confirm the predominant C-glycosylation pathway, enzymatic properties, primary amino acid sequence and gene sequence of CGT and to provide a good foundation for further studying CGT from P. edulis leaves.[Method] The CGT of a C-glycosyl flavonoid from P. edulis leaves was first purified by ammonium sulfate fractionation precipitation, dialysis, gel chromatography and diethylaminoethyl anion exchange, and detected using SDS-PAGE. Three possible substrates (eriodictyol chalcone, eriodictyol and luteolin) were selected as substrates to analyse the possible C-glycosylation pathways using the established C-glycosylation reaction system and to confirm which pathway was predominant. Using Q-TOF analysis and database searching, the gene and amino acid sequences of P. edulis CGT were analysed and confirmed.[Result] The results suggested that the molecular weight of P. edulis CGT is about 50 kDa, and the optimal incubation time, incubation temperature, buffer pH and substrate (luteolin) concentration for the CGT reaction system was 40 min, 28 ℃, 8.1 and 31.76 μmol·L^-1, respectively. A large amount of the luteolin underwent CGT-catalysed C-glycosylation was converted into isoorientin in reaction system 3, while a large amount of eriodictyol chalcone and eriodictyol in reaction system 1 and 2 were converted into unknown products, only a small portion was converted into isoorientin. According to fragmenting law and the diagnostic fragments of the protein peptide ions, four CGT peptide ions were analysed and confirmed, which was aligned with the amino acid sequence encoded by the gene from P. edulis gene (PH01000603G0510), the amino acid sequence of the four CGT peptide ions perfectly matched the amino acid sequence encoded by the P. edulis gene (PH01000603G0510). The matching rate of P. edulis gene sequence (PH01000603G0510) with rice (Oryza sativa Japonica Group) CGT gene sequence (PH01000603G0510) was 81%.[Conclusion] Through separation, purification and structural identification of CGT from P. edulis, the possible gene (PH01000603G0510) and amino acid sequences of C-glycosyl flavonoid CGT from P. edulis leaves were analysed and confirmed. The C-glycosylation pathways of flavonoids in P. edulis were also determined. The main C-glycosylation pathway of isoorientin in P. edulis leaves is via the direct synthesis of isoorientin from luteolin and UDP-glucose, and the secondary pathway is via the indirect synthesis of isoorientin from eriodictyol chalcone and UDP-glucose, or eriodictyol and UDP-glucose. The C-glycosyl flavonoid CGT in P. edulis leaves catalyzes the accumulation of flavone 6-C-glucosides (isoorientin) but seldom catalyzes the accumulation of flavone 8-C-glucosides (orientin).

Key words: Phyllostachys edulis, C-glycosyltransferase, C-glycosylation, isoorientin, luteolin