2.8. Quantitative RT-PCR

True leaf tissue samples were collected from four-week-old seedlings at the indicated time points after DEX spray and used for detecting transcripts of AC transgene. For all other genes, aerial part tissue (excluding cotyledons) samples from five-week-old seedlings were used. Total RNA was isolated using the Spin Column Plant Total RNA Purification Kit (Sangon Biotech, Shanghai, China). After DNase I digestion, total RNA was subjected to reverse transcription (RT) using the HIScript® III RT SuperMix for qPCR (+ gDNA wiper) Reagent Kit (Vazyme Biotech, Nanjing, China). The ChamQ^TM Universal SYBR® qPCR Master Mix (Vazyme Biotech) was used to prepare PCR reaction mixture for amplification on LightCycler® 480 II Real-Time PCR System (Roche, Basel, Switzerland) with the following conditions: 30 s at 95°C, 40 cycles of 95°C for 10 s, and 60°C for 30 s. All primers used in this study are listed in Table S1. Melting curve analysis and agarose gel electrophoresis were performed to verify the specificity of amplified products. Relative expression levels were normalized to an internal control gene F-box and calculated using the 2^–∆∆Cq method. F-box has been verified by systemic studies in Brassica napus to serve as an optimal reference gene for quantitative RT-PCR normalization in a previous publication.³⁶ In addition, independent amplification experiments with F-box showed its stable expression in the samples under control conditions and DEX treatment, which further validated the suitability of F-box for the analyses (Table S2). Three biological replicates were analyzed in all these experiments.

The sequences of Brassica napus genes were obtained from the Bra_napus_v2.0 genome assembly of cultivar ZS11 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000686985.2/).