Sybr green master mix reagent

Total moso bamboo leaf RNA under different stresses, and from different organs, was extracted with TRIzol reagent (Invitrogen, NH, USA) according to the manufacturer’s instructions. The primer pairs for the 18 selected PhebZIP genes were designed using Primer Express 3.0, and the specificity of primers was checked using BLAST searches against a coding sequence (CDS) database downloaded from the moso bamboo genome database (http://server.ncgr.ac.cn/bamboo/index.php) (access in January 2019). [46 (link)]. The tonoplast intrinsic protein 41 (TIP41) gene, which is stably expressed in moso bamboo, was used as a reference to normalize expression data [62 (link)]. The qRT-PCR amplifications using SYBR Green Master Mix reagent (Applied Biosystems) were performed on an ABI 7300 Real-Time system (Applied Biosystems) in 20 μL reactions containing 1 μL of each gene-specific primer, 1 μL of cDNA sample, 7 μL ddH₂O, and 10 μL SYBR Green Master Mix reagent (Applied Biosystems) [63 (link)]. All primers for amplification of PhebZIP genes are given in Table S5. The qRT-PCR amplification conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 55 ° C for 15 s, and 72 °C for 10 s. A melting curve analysis was performed to determine those primers with the highest specificity and efficiency. The relative expression levels were assessed based on the 2^−ΔΔCT method [64 (link)].

qRT-PCR was used to validate the RNA-Seq results on 20 randomly selected gene accessions. The β-actin gene was used as an internal reference, and primers were designed using Primer5 software (Table S2). RNA samples from FW and SM (with three replicates per group) were used for qRT-PCR. First-strand cDNA was synthesized by SuperScript™ III First-strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocols. Briefly, qRT-PCR was performed in the optical 96-well plates with an ABI PRISM 7500 Real-time Detection System (Life Technology, Grand Island, NY, USA). The amplification was performed in a total volume of 15 μL, containing 7.5 μL 2× SYBR Green Master Mix reagent (Life Technology), 1 μL of cDNA (100 ng/μL), and 0.3 μL of 10 μM of each gene-specific primer. PCR was conducted as follows: 50 °C for 2 min, 95 °C for 10 min, 45 cycles of 95 °C for 15 s, and 60 °C for 1 min. After PCR, data were analyzed with ABI 7500 SDS software. The comparative Ct method (2^−∆∆Ct method) was used to analyze target gene expression. All data are shown at levels relative to the expression of the β-actin gene.

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.