MeJA-Induced Transcriptional Analysis in Wolfiporia cocos

Total RNA was extracted from 10-day-old W. cocos mycelia under the treatment of 200 μM MeJA for 0 h (CK), 2 h (T2), and 12 h (T12), respectively. The RNA was then treated with recombinant DNase I (Life Technologies, Burlington, Canada) at a concentration of 1.5 U/μg total RNA. Single-strand cDNA was synthesized using the PrimeScript™ 1^st Strand cDNA Synthesis kit (TaKaRa, Shlga, Japan), 1 μg RNase-free DNase I-treated (TaKaRa) total RNA, and random primers. RT-qPCR was performed at least three times for each sequence using SYBR®Premix Ex TaqTM (Perfect Real Time) (TaKaRa) and an ABI PRISM 7500 real-time PCR System (Life Technologies). Each reaction contained 7.5 μl 2× SYBR Green Master Mix Reagent (Life Technologies), 1.0 μl (10 ng) cDNA, and 200 nM gene-specific primers in a total reaction volume of 15 μl. The PCR amplification program consisted of denaturation at 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. The relative gene expression data were normalized against an internal reference gene, cyclophilin (CYP) [23] (link). The relative expression levels were calculated by the 2^−ΔΔCt method. The primers were designed using Primer3 (http://frodo.wi.mit.edu/primer3/). The primers used for RT-qPCR in this study are listed in Table S35.