2.3 Quantitative real-time PCR
The total DNA was extracted from A. alternata (WT) andAaFla1 OE using a fungal RNA extraction kit
(CWBIO, Jiangsu, China). After extraction, the total RNA quality and
concentration were measured and DNA was removed, and reverse transcribed
into cDNA with reference to the GenStar kit (Genstar, Bei-jing, China).
Using a two-step amplification, the quantitative real-time PCR (qPCR)
method was pre-denatured at 95°C for 5 min, denatured at 95°C for 30 s,
denatured at 56°C for 30 s, and extended at 72°C for 45 s, 40 cycles.
The β-Actin gene was used as an internal reference gene (Table S1).
After completion of the reaction, the expression was calculated
according to the 2-△△CT method to analyze the
transcript levels of the genes. All experiments were repeated three
times.