Agrobacterium tumefaciens–mediated genetic transformation and overexpression of the flavonoid 3′5′-hydroxylase gene increases the flavonoid content of the transgenic Aconitum carmichaelii Debx. plant

Aconitum carmichaelii Debx. is a medicinal plant that contains a variety of valuable medicinal substances, including flavonoids, alkaloids, and polysaccharides. Flavonoids are important substances because of their antioxidant, antiproliferative, and anticancer effects, but the flavonoid content in A. carmichaelii is low. Flavonoid 3′5′-hydroxylase (F3′5′H) is a key enzyme that catalyzes the final reactions in flavonoid biosynthesis in A. carmichaelii. Therefore, overexpressing the gene encoding of the F3′5′H enzyme to increase the flavonoid content in A. carmichaelii is a new direction in medicinal plant research strategies. In this work, the results of Agrobacterium tumefaciens–mediated genetic transformation of uidA and AcF3′5′H in A. carmichaelii plants, expression analysis of AcF3′5′H, and production of transgenic A. carmichaelii plants have been presented. Out of 180 transformed samples, 24 transgenic A. carmichaelii plants developed normally under greenhouse conditions. The results of PCR analysis of 15 transgenic A. carmichaelii plants showed that eight transgenic plants contained the AcF3′5′H transgene. Using electrophoresis SDS-PAGE, western blot, and ELISA techniques, we identified the expression of AcF3′5′H protein (rAcF3′5′H) in three transgenic plants. The rAcF3′5′H protein content of transgenic plants ranged from 0.2083 μg μL⁻¹ (transgenic line T₀-6) to 0.2507 μg μL⁻¹ (T₀-4). The highest concentrations of flavonoids, 773.50 ± 12.87, 661.73 ± 2.85, and 761.61 ± 9.10 μg g⁻¹, were found in transgenic lines T₀-4, T₀-6, and T₀-13, respectively. These values were higher than those of the non-transformed plants (wild-type). Our results demonstrate that compared with non-transgenic plants, overexpression of AcF3′5′H significantly increased the AcF3′5′H protein content in transgenic plants and the total flavonoid content in transgenic lines T₀-4, T₀-6, T₀-13, from 39.13 to 63.63%. This is the first report of genetic transformation and an analysis of the expression enhancement of F3′5′H in the A. carmichaelii plant.