2.1 Plant materials and growth conditions
The Honghua Dajinyua cultivar (N. tabacum ) kept by our lab was used for transgenic manipulation, gene expression analysis, and detection of flavonoids content in the present study. Tobacco seeds were soaked and sterilized as described previously (Z. Wang et al., 2018; Z. Wang et al., 2020), and then germinated on MS medium. The sterile seedlings were either used for transgenic manipulation, or transplanted into pots for normal growth (28°C/16h light, 23°C/8h darkness) until the flowering stage. Various tissues were collected from the plants for RNA extraction or flavonoids content detection.