*4.3. RNA Isolation and Real-Time PCR Analysis*

Total RNA was extracted using a Trizol Reagent kit (Invitrogen, CA, USA) according to the manufacturer's instructions and analyzed by gel electrophoresis. The first-strand cDNA was synthesized with random oligonucleotides using the HiScript® II Reverse Transcriptase system (Vazyme, Nanjing, China). qRT-PCR was carried out in a total volume of 20 µL containing 2 µL of cDNA, 0.4 µL gene-specific primers (10 µM), 10 µL SYBR Green Mix and 7.2 µL of RNase free ddH2O, using the Roche LightCycler480 Real-time System (Roche, Basel, Swiss Confederation). The expression was represented in the form of relative fold change using the 2−∆∆*C*<sup>T</sup> method [79]. Differentially expressed genes between each two samples pair were defined as two-fold up-regulated or two-fold down-regulated genes. Primers used for qRT-PCR are designed by Primer3 (Table S3). Three biological replications were performed. Heat map analysis of the expression data was performed using heat map drawing software MeV (version No. 4.7, Institute for Genomic Research, MD, USA)
