In experiments on the effect of light stimulation on mycelial growth and in gene expression analysis, colonies about 65 mm in diameter grown on the GPY agar media at 20oC in the dark were used. An ELUX-1096 LED lighting unit for plant cultivation research (CCS, Kyoto, Japan) was used for the culture of mycelia, and the temperature was kept constant at 20oC in the dark. Light intensity was set using photon flax density (PFD) measurements taken with an LI-190 quantum sensor (LI-COR Bioscience, Lincoln, NE). Mycelial colonies about 65 mm in diameter grown on GPY agar media at 20oC in the dark were irradiated at 15 oC for 0.5 – 36 h using blue LED at 150 μmol m-2 s-1 of PFD for the analysis of gene expression in the mycelia. The colonies irradiated were then immediately frozen in liquid nitrogen and stored at -80 ºC until use.
Growth protocol
Modified MA medium consisted of 10 g of malt extract, 10 g of D(+)-glucose, 4 g of yeast extract, and 25 g of agar in 1 liter of distilled water. GPY medium consisted of 50 g of D(+)-glucose, 2.5 g of polypeptone, 1.0 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.5 g of CaCl2 2H2O, 10 mg of FeCl2 6H2O, 7.2 mg of MnCl2 4H2O, 4.0 mg of ZnCl2, 1.0 mg of CuSO4 5H2O, 2.5 g of yeast extract, and 25 g of agar in 1 liter of distilled water. The initial pH was adjusted to 5.5. The media were autoclaved at 121oC for 20 min before use. The oyster mushroom KH-3 dikaryotic strain was first incubated at 20oC in the dark on an MA medium in a Pyrex Petri dish (diameter, 9 cm). The fungal thread was grown concentrically to form a mycelial colony about 70 mm in diameter. From the periphery of the mycelial colony, a colony 6 mm in diameter was cut out and inoculated at the center of a GPY medium in a Petri dish.
Extracted molecule
total RNA
Extraction protocol
Total RNAs were extracted from ca. 50 - 100 mg of the frozen colonies with an RNeasy Plant Mini Kit (Qiagen, Tokyo) following the manufacturer’s instructions. Contaminating genomic DNA was removed using an RNase-Free DNase Set (Qiagen). The quantity and quality of total RNAs was evaluated with a Nanodrop ND-1000 spectrophotometer (Themo Fisher Scientific Inc., Waltham, MA) and an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA), as recommended.
Label
Cy3
Label protocol
Total RNA was amplified and labelled with Cyanine 3 (Cy3) using Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies, Palo Alto, CA) following the manufacture’s instructions. After amplification and labeling, cRNA quantity and cyanine incorporation were determined using a Nanodrop ND-100 spectrophotometer and an Agilent Bioanlyzer 2100.
Hybridization protocol
For each hybridization, 1.65 mg of Cy3 labeled cRNA was fragmented, and hybridized at 65 oC for 17 h to an Agilent GE8x15K custom microarray.
Scan protocol
After washing, microarrays were scanned using an Agilent DNA microarray scanner.
Data processing
Intensity values of each scanned feature were quantified using Agilent feature extraction software ver. 10.7.3.1, which performs background subtractions. We only used features which were flagged as no errors (present flags) and excluded features which were not positive, not significant, not uniform, not above background, saturated, and population outlines (marginal and absent flags). Normalization was performed using Agilent GeneSpring GX ver. 11.0.2 (per chip: normalization to 75 percentile shift; per gene: normalization to median of all samples). There are total of 12,244 probes on an Agilent GE8x15K custom microarray without control probes. The altered transcripts were quantified using the comparative method. In this study we applied more than or equal to 2-fold change in signal intensity to identify the significant differences of gene expression. The differentially expressed genes were also subjected to K-means analysis using GeneSpring ver. 11.0.2 software.
