Transgenic plasmid construction and plant transformation

To confirm the function of NnMYB5 gene in lotus, the genomic DNA of NnMYB5 was amplified and cloned into the pCAMBIA1301 vector under the control of CaMV 35S promoter with the primers 5′-CGAGCTCATGGATGGTGGTTTGGGTTT-3′ (forward) and 5′-CGGGATCCTCAATAACTCCACCACCTATG-3′ (reverse).

The above recombinant construct was then transformed into Agrobacterium tumefaciens strain GV3101 and introduced into Arabidopsis thaliana WT (Col-0) plants using the floral dip method (Clough & Bent, 1998) to obtain NnMYB5 over-expressing plants. Arabidopsis seeds were sterilized with 70% (v/v) ethyl alcohol, 10% (w/v) NaCl and deionized water. After stratification at 4 °C for 3 d in darkness, transgenic Arabidopsis seeds were selected on half-strength Murashige and Skoog (MS) media containing 30 mg/L Hygromycin. Hygromycin-resistant T1 seedlings were transferred to soil and grown at 23 °C in a growth chamber with a 16-h day length. T2 seedlings were sowed in soil. The nutrient solution was watered from below in pots with soil-grown plants twice every week. The transgenic plants were confirmed by PCR analyses. Additionally, the expression levels of NnMYB5 and TT19 (At5g17220) TT19 gene were determined by semi-quantitative RT-PCR (sqRT-PCR) and the actin gene was used as an internal control. The primers used for sqRT-PCR are shown (Table S2).