Liquid spawn preparation
Oyster mushroom (P. ostreatus) strain zaoqiu615 was selected because it is a stable commercial strain available in Jilin Province, China. The stock culture of strain zaoqiu615 was maintained on potato dextrose agar (PDA) slants at 25 °C for 7 days. Liquid spawn was prepared by culturing the mycelia in a liquid medium at 23.8 °C for 7 days. The liquid medium (neutral pH) consisted of 100 g/L potato, 15 g/L brown sugar, 10 g/L glucose, 45 g/L wheat bran, 2.5 g/L peptone, 2.0 g/L KH2PO4, 1 g/L MgSO4, 10 mg/L vitamin B1, and 0.3 mL/L glycerol.
Substrates preparation and light treatment
The liquid spawn was inoculated into sterilized bags (17 cm × 33 cm) containing the growth medium (purchased from the Mushroom base of Jilin Agricultural University, Changchun, China) for radial mycelial growth and fruiting body cultivation. The growth medium (65% moisture content) consisted of corn cob (24%), wood shavings (35%), wheat bran (24%), corn flour (10%), soybean meal (5%), temperament calcium carbonate (1%), and lime (1%). The bags were placed in a mushroom incubator (Hipoint Corporation, Taiwan) at 24 °C, 55% air moisture, and dark condition for 20 days. After 20 days of inoculation, P. ostreatus primordium emerged. At this stage, the bags containing newly emerged primordium were subjected to blue-light, red-light, and dark treatments at 24 °C for 7 days. Blue-light and red-light treatments were conducted using an LED blue lighting unit (430–470 nm) and LED red lighting unit (610–640 nm) in the mushroom incubator (MI302, Hipoint Corporation), respectively. The light intensity was about 50 μmol/m2/S. After 7 days of light treatment, the stipe, pileus, and gill of each mushroom were collected for RNA extraction. Fresh weights of the stipe and pileus + gill for each treatment were determined, with 10 mushrooms for each treatment.
Each treatment and each tissue comprised three biological replicates, and each biological replicate consisted of three mushrooms. A total of 2 μg of RNA per sample were used as the input material for RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (#E7530L, NEB, USA) following the manufacturer’s recommendations. In brief, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was conducted using divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5 ×) under elevated temperature. First-strand cDNA was synthesized using random hexamer primer and RNase H, and second-strand cDNA synthesis was subsequently performed using dNTPs, DNA polymerase I, and RNase H. The library fragments were purified using QiaQuick PCR kits, and eluted with EB buffer. Then, terminal repair, A-tailing, and adapter were implemented. The libraries were sequenced on an Illumina platform and 150-bp paired-end reads were generated. Finally, about 6 Gb of clean data was obtained for each sample. The genome sequence of P. ostreatus PC15 strain was used as a reference genome (NCBI accession PRJNA81933, Riley et al. 2014). The obtained clean data were aligned to the reference genome using HISAT2 v2.1.0, the successor to TopHat2, which uses a modified BWT algorithm to convert reference genomes to index at a faster speed and with fewer resources. The FPKM value was used to define the expression level of each gene, and DESeq 2 was employed to determine the differentially expressed genes (DEGs) among treatments (adjusted P ≤ 0.05 and |log2fold change | ≥ 1). The number of DEGs used to make a Venn diagram was ascertained, and the fold change values of all genes were subjected to KEGG enrichment analysis using the clusterProfiler R package (GSEA method) (Yu et al. 2012). The generated P values were adjusted using the BH method (Yu et al. 2012).
Quantitative real-time PCR
Each treatment and each tissue comprised three biological replicates, and each biological replicate consisted of three mushrooms. The total RNA from each organ of the mushroom was extracted using TRIzol reagent (Invitrogen). The RNA was treated with DNaseI (Invitrogen), reverse-transcribed using SuperScriptTM RNase H-Reverse Transcriptase (Invitrogen), and then subjected to quantitative real-time PCR (qRT-PCR) using gene-specific primers (Additional file 1: Table S1 online). Amplification of the target gene during each cycle was monitored by using SYBR Green, and Actin (ID gene_9983) and GPDH (gene_10642) mRNA amplifications were used as internal quantitative control. The relative expression of the target genes was calculated using the △△Ct method (Livak and Schmittgen 2001).
Differences in fresh weight and qRT-PCR results was determined by t test using SPSS version 16.0 (IBM). Statistical significance was set at P < 0.05. The statistical test for RNA-Seq data was performed using the DESeq 2 R package.