Plant RNA Isolation and RT-qPCR Analysis

RNA was isolated using the EASYspin Plus Plant Quick RNA isolation Kit (RN38) (Aidlab Biotechnology, Beijing, China) according to the manufacturer’s instructions. The first strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). The primers of RT-qPCR were designed by BatchPrimer3v1.0 (http://batchprimer3.bioinformatics.ucdavis.edu/index.html) and the Actin (1) reference gene was used as the internal control [49 (link)]. The primers used for RT-qPCR are listed in Table S1. RT-qPCR was carried out using a LightCycler 480Ⅱ real-time PCR system (Roche, Switzerland) using the RealUniversal Color PreMix (SYBR Green) (TIANGEN, Beijing, China) according to the manufacturer’s instructions. Three biological replicates were performed for each sample. The relative expression levels were calculated using the comparative 2^−△△CT method [50 (link)].