RNA extraction and quantitative Real-Time reverse transcription polymerase chain reaction (qRT-PCR) analysis

Total RNAs were extracted from leaves using RNAiso plus (Takara, Japan). The cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransScript, China) with Oligo-dT18 primer. qRT-PCR analysis followed the guidelines and protocols described previously [51]. All reactions were performed using the SYBR Premix (5.0 µL of 2× SYBR Premix Go Taq II, 0.5 µL of primers, 1.0 µL of cDNA, and 3.5 µL of ddH2O). Melting curve analysis of qRT-PCR samples revealed that there was only one product for each gene primer reaction. The PCR products were sequenced to confirm the specific amplification. A house-keeping gene BjEF-1-α37 was used as an internal standard in tissues. The relative expression levels were counted using the formula 2−△△Cq as described in Bio-Rad protocol, and statistical differences were calculated using student’s test. Three biological replications and three technical replications were performed in qRT-PCR. The primers used in qRT-PCR analysis were shown in Table S3.

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