The effect of RNAi-induced silencing of FaDFR on anthocyanin metabolism in strawberry (Fragaria × ananassa) fruit

• RNAi in this study was finished through instantaneous conversion, laying the foundation for the complete conversion to gain a transgenic strawberry for the future.
• FaDFR is one of the key genes in the anthocyanin biosynthesis pathway in strawberry fruit.
• Metabolites were shunted to the quercetin-glycoside biosynthesis pathway after silencing of FaDFR.

Dihydroflavonol 4-reductase (DFR) converts dihydroflavonol into leucoanthocyanidin in the anthocyanin biosynthetic pathway. In this study, we cloned FaDFR from strawberry (Fragaria × ananassa) fruit by RT-PCR; the cDNA was 1025 bp in length and encoded a predicted protein of 340 amino acids. Sense and anti-sense fragments amplified from the FaDFR coding sequence (CDS) were used to construct pBI-DFRi, a plant expression vector containing an RNAi silencing cassette. Agrobacterium strain GV3101 carrying pBI-DFRi was evenly injected throughout the entire fruit while it was still attached to the plant about 14 days after pollination. The phenotype was observed 14 days post-injection, and fruits were analyzed by semi-quantitative RT-PCR and HPLC-MS analysis. The results showed that FaDFR was down-regulated and fruit color became pale in the agroinfiltrated fruits compared to controls, and there was an accompanying change in anthocyanin concentration. Pelargonidin-glycosides, cyanidin-glycosides and kaempferol-glycosides decreased by 93.3%, 97.2%, and 55.6% in the pB1-DFRi infiltrated fruits compared with controls, indicating that FaDFR is one of the key genes in the anthocyanin biosynthesis pathway in strawberry fruit. In contrast, quercetin-glycosides increased by 73.1%, and we conclude that metabolites were shunted to the quercetin-glycoside biosynthesis pathway when the anthocyanin biosynthesis pathway was blocked.