Functional Analysis Of Dihydroflavonol 4-reductase (DFR) Gene From Orange-flower Gentian (Gentian Lutea L.var.aurantiaca)

Anthocyanins are important pigments for flower color of higher plants which is an important factor determining the appreciation value and economic value of floriculture plants.Dihydroflavonol 4-reductase(DFR),which catalyzes the reduction of dihydroflavonols to leucoanthocyanins,is a key enzyme in the biosynthesis of anthocyanidins.DFRs from several plants can catalyze the reduction of three dihydroflavonols,kaempferol(DHK),dihydromyricetin(DHM)and dihydroquercetin(DHQ)into leucoanthocyanidins,which are common precursors for anthocyanin and CTs(proanthocyanidins)synthesis.Thus,DFR is an important regulatory point for the formation of anthocyanins,which plays an important role in the formation of flower color.GlaDFR1 and GlaDFR2 cDNAs were isolated from the petals of orange-flowered gentian(Gentiana lutea L.var.aurantiaca)by our laboratory.Sequence analyses indicate that both genes contain a full-length open reading frame encoding 374 amino acids.DFRs belong to NADP(H)dependence members of the family of short chain reductase(SDR)and both DFRs have NADP(H)and substrate domain.Meanwhile,we also found that GlaDFR1 and GlaDFR2 belong to Asp-type DFR.To investigate the function of the GlaDFR1 and GlaDFR2,the plant expression vectors(pCAMBIA1302-GlaDFR1 and pCAMBIA1302-GlaDFR2)for both genes under the control of the cauliflower mosaic virus(CaMV)35S promoter were constructed.These two expression vectors were transferred into Agrobacterium tumefaciens(EHA105),respectively.GlaDFR1 and GlaDFR2 genes were transformed into Nicotiana tabacum genome by Agrobacterium mediated leaf disc transformation.Molecular characterization and phenotype analysis of T1 transgenic tobacco plants were performed.To identify transformed plants carrying integrated DNA,genomic PCR was carried out to amplify the target genes from genomic DNA of transgenic tobacco plants.The results have shown that the target genes(GlaDFR1 and GlaDFR2)was successfully inserted into the genome of transgenic plants.Semiquantitive RT-PCR was performed to monitor the expression of GlaDFR1 and GlaDFR2.Results indicated that GlaDFR1 and GlaDFR2 could be transcribed.The flowers of the transgenic tobacco expressing GlaDFRs had distinct pigment differences from wild-type plants.The petals of wild type tobacco show pink color whereas the petals of transgenic tobacco were dark pink color.Anthocyanidin analysis using High Performance Liquid Chromatography(HPLC)showed that cyanidin derivatives were reduced in the petals of transgenic lines as comapred to those of untransformed tobacco.These data revealed that GlaDFR1 and GlaDFR2 have the function of dihydroflavonol 4-reductase and participate in anthocyanin of synthesis.