qRT-PCR Analysis of the Nox Gene in T. rubrum

The suspension of T. rubrum was sampled and ground in liquid nitrogen. Total RNA was extracted using a HiPure Fungal RNA Kit (Magen, Guangzhou, China) following the manufacturer’s instructions. The extracted RNA was treated using a DNase On Column Kit (Magen, Guangzhou, China) at 37°C for 30 min to remove the genomic DNA. Subsequently, a PrimeScript^TM RT Master Mix Kit (Takara, Dalian, China) was used for reverse transcription. β-Tubulin of T. rubrum was selected as the reference gene. The gene-specific primers were designed using Primer 5.0 Software. For β-tubulin, the primers were β-tubulin-FP, 5′-CCGCTCTTTGCTCTATTCCT-3′, and β-tubulin-RP, 5′-CCATCTCGTCCATACCCTCA-3′. For NADPH oxidase (Nox), the primers were Nox-FP, 5′-TGGCTGTGACTTTGACGAGA-3′, and Nox-RP, 5′-CCGACTAACACCGCTACTTC-3′. The qRT-PCR experiment was conducted using a SYBR Premix Ex Taq^TM Kit (Takara, Dalian, China) and a LightCycler^® 480 II system (Roche, Mannheim, Germany). All values were normalized to those of β-tubulin expression. Relative expression was analyzed using the comparative Ct method (2^-ΔΔC_T). A probability (p) value ≤ 0.05 was considered to indicate significance differences. (Only strain ATCC4438 was used in the qRT-PCR analysis.)