Functional Characterization Of Dihydroflavonol-4-Reductase Gene From Freesia Hybrida

Freesia hybrida is a good material for studying the secondary metabolism of flower color because of the freesias has the range of flower colours available.Anthocyanidin belong to the secondary metabolism substance of plant and also is the dominant factor to affect the flower color.Dihydroflavonol 4-reductase(DFR),which catalyzes the reduction of dihydroflavonols to leucoanthocyanins,is a key enzyme in the biosynthesis of anthocyanidins.DFR preparations from several plants can catalyze the reduction of three dihydroflavonols,kaempferol(DHK),dihydroquercetin(DHQ)and dihydromyricetin(DHM)into leucoanthocyanidins,which are common precursors for anthocyanin and condensed tannin synthesis.Five FhDFRs full-length cDNA of FhDFR1,FhDFR4,FhDFR5,FhDFR7 and FhDFR8 were obtained from the F.hybrida using the SMART TM RACE cDNA Amplification Kit.Sequence analysis results that they have a full-length open reading frame encoding 304 ~ 357 amino acids.DFR belonged to NADP(H)dependence members of the family of short chain reductase(SDR)by comparing of the DFR from F.hybrida and other plant,and indicated DFR have NADP(H)and substrate domain.Moreover,We also discovered that FhDFR2 and FhDFR6 belong to the Asn-type DFR,while FhDFR3 belong to the Asp-type DFR.The light quality and illumination time affects expression quantity of FhDFRs was studied with Real time-qPCR.Real time-qPCR analysis showed that the eight FhDFRs genes have different expression patterns.The suggest that the DFR from F.hybrida gene family has occurred function disproportionation the course of evolution.Our previous study showed that the expression of FhDFR2,FhDFR3 and FhDFR6 genes a significant related to anthocyanin accumulate,and FhDFRs have high homology with the DFRs from other plant species.To investigate the function of the FhDFRs in the F.hybrida anthocyanins and observe the transgenic Arabidopsis recovery of missing phenotype,transgenic Arabidopsis mutants plants that expressed FhDFRs genes under the control of the 35 S promoter were produced.Meanwhile,we subcloned the coding regions of FhDFRs into the pET28 a by T7 promoter and expressed in prokaryote.Soluble protein extracts from IPTG-induced cels were prepared and assayed for enzyme activity using High Performance Liquid Chromatography(HPLC).Results shown that transgenic plants harboring the 35S:FhDFR2,35S:FhDFR3,35S:FhDFR6 transcription cassette produced much darker purple than were observed on Arabidopsis mutants control plants and accompanied by anthocyanins content in part of the recovery.FhDFR2,FhDFR3,FhDFR6 all can reduce dihydromyricetin,including FhDFR2 also reduce dihydroquercetin but all have not reduce dihydrokaempferol.These data revealed that FhDFR2,FhDFR3,FhDFR6 have the function of dihydroflavonol 4-reductase,participate in anthocyanin of F.hybrida synthesis.In our previous study that red flowers of F.hybrida anthocyanins is primarily a delphinium derivatives and little cyanidin derivatives,but does not contain pelargonidin derivatives.So presumably that FhDFR gene has played a decisive role in the F.hybrida anthocyanins biosynthesis pathway.