Cloning and functional analysis of Rhododendron agastum anthocyanidin reductase gene RaANR1

【Objective】 The aim of this study was to clone Rhododendron agastum anthocyanidin reductase （ANR） gene RaANR1， analyze its regulatory function in the biosynthesis of anthocyanidins and proanthocyanidins， and provide theoretical basis for the genetic improvement of flower color in Rhododendron agastum and the application of RaANR1 gene in plant metabolic engineering.【Method】 RaANR1 gene was cloned using Rhododendron agastum flowers as material. Bioinformatics analysis on RaANR1 gene was performed using relevant softwares. RaANR1 was transfected into wild-type Arabidopsis thaliana and tobacco by dipping method and injection method respectively. After resistance screening and reverse transcription PCR （RT-PCR） verification，anthocyanin and proanthocyanidin contents in transgenic Arabidopsis thaliana seedlings and tobacco flowers were detected using spectrophotometer. The relative expression of endogenous genes in the synthesis of anthocyanins and proanthocyanidins in tobacco was also detected by real-time fluorescence quantitative PCR.【Result】 RaANR1 gene was successfully cloned. Its coding region （CDS） was 1002 bp，encoding 333 amino acids. The encoded protein was a stable， non-secretory and hydrophilic protein. In its secondary structure，α-helices and random coils were the most abundant， and it contained the conserved NADPH/NADP binding domain，active site and substrate-binding site of ANR. The amino acid sequence similarities of RaANR1 with Camellia sinensis CsANR，Diospyros kaki DkANR and Vaccinium ashei VaANR were 82.49%，80.88%，and 89.79% respectively. RaANR1 was most closely related to VaANR and they shared the most similar conserved motifs. Compared with wild-type Arabidopsis thaliana， seedlings of the two transgenic Arabidopsis thaliana lines exhibited a color shift from purple to green in both cotyledons and hypocotyls， and the anthocyanin content was significantly lower than that of wild-type plants （P<0.001）， whereas the proanthocyanidin content in the two transgenic lines was significantly （P<0.05， the same below） or extremely significantly （P<0.001） higher than wild-type plants. In two transgenic tobacco lines， flower color was distinctly paler than that of the wild-type tobacco， and anthocyanin content in flowers were extremely significantly reduced （P<0.001， P<0.01） compared to wild-type tobacco， while proanthocyanidin content was extremely significantly （P<0.001） or significantly increased. Compared with the wild-type tobacco， the expression levels of NtF3'5'H， NtDFR， NtANS， and NtANR1 genes were significantly up-regulated in the two transgenic lines， whereas no significant difference was observed for NtF3'H and NtANR2 genes （P>0.05）.【Conclusion】 Overexpression of RaANR1 gene drastically decreases anthocyanin content in Arabidopsis thaliana seedling cotyledons and tobacco flowers， while significantly promotes proanthocyanidin content. These results indicate that RaANR1 gene modulates flower color by regulating the synthesis of proanthocyanidins and anthocyanidins.