Quantitative RT-PCR Analysis of Gene Expression

Total RNA from the different tissues and leaf axils was extracted with an RNAprep Pure Plant Kit (TIANGEN, Beijing, China) according to the kit protocol, and DNA contamination was removed with RNase-free DNase I. First-strand cDNA was synthesized with a Hifair^®Ⅱ 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China) according to the kit protocol. Gene-specific primers for qRT-PCR were designed with Primer 6.0 (Supplemental Table S2). The LilyActin primer was used as an internal control [66 (link)], and SYBR^® Green Master Mix (No Rox) (Yeasen, Shanghai, China) was used in the reaction mixture according to the manufacturer’s instructions. qRT-PCR was conducted using the CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA), with an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 72 °C for 1 min. A melting curve analysis was performed for each primer pair to confirm its specificity. The 2^−ΔΔCt method was used to calculate the relative expression levels of the different genes [67 (link)]. Three biological and three technical replicates were used to reduce error.