Cloning and Expression of Dihydroflavonol 4-Reductase in Actinidia chinensis var. rufopulpa

Two DFR cDNA sequences similar to full-length DFR cDNA in Vitis vinifera were reconstructed by overlapping ESTs from Actinidia dbEST.Based on these two sequences,a full-length AcDFR1(1264 bp) and partial sequence of AcDFR2 cDNA near 3’ end(880 bp) was obtained using 3’RACE and 5’RACE in A. chinensis var.rufopulpa.AcDFR1,whose nucleotide sequence exhibited 84% identity with DFR cDNA of Camellia sinensis,showed 80% identity with Gerbera hybrida var.regina at the amino acid level.Alignment analysis of DFR showed that AcDFR1 differed considerably from AcDFR2.To understand the diversity of expression among green,yellow,and red-fleshed cultivars,real-time PCR was conducted.AcDFR1 expression was extremely high in green-flesh kiwifruit(Jinkui) but relatively low in the yellow-flesh fruit(Jinnong) and red-flesh fruit(Hongyang).There was an increasingly higher expression of AcDFR1 and AcDFR2 in Jinnong and Hongyang fruit after development,especially between 90 to 120 days after flowering.This indicated that the two transcriptions both catalyzed the accumulation of anthocyanins during fruit growing development.Results also suggested that AcDFR1 and AcDFR2 both participated in the anthocyanin pathway.In addition,AcDFR1 may have additional functions related to other flavonol metallization referred to upper-stream pathways of Jinnong and Jinkui fruit.