Sybr premix ex taq 2

qRT‐PCR was performed using an ABI7500 real‐time PCR system (Applied Biosystems, USA). cDNA was amplified using the SYBR Premix Ex Taq II (TaKaRa, Japan) according to the manufacturer's protocol. Reactions were prepared in a total volume of 10 μl containing 1 μl diluted cDNA (100 ng/μl), 5 μl 2 × SYBR Premix ExTaq II (TaKaRa, Japan), 3 μl RNase‐free sterile water, and 0.5 μl each of the forward and reverse primers (10 ng/μl). The PCR program was 95°C for 30 s, 40 cycles of 95°C for 5 s, 55°C for 30 s, and 72°C for 30 s, followed by a melting curve analysis to confirm the specificity of amplification for each reaction. The reaction solution without a cDNA template was used as a negative control to confirm template‐specific amplification. PCR reactions were conducted for three biological replicates, and the detection of each gene was performed in an independent sample with three technical replicates.

Total cellular mRNA was isolated using Trizol agent (Invitrogen), following the manufacturer’s instructions. The residual genomic DNA was wiped off prior to cDNA synthesis using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara) on Gene amplification Machine (Gene Touch, BIOER, HangZhou, China) according to the manufacturer’s instructions. Quantitative reverse transcriptase PCR (qRT-PCR) was performed using 20 μl reaction volume containing 10 μl 2 × SYBR Premix Ex Taq II (SYBR^® Premix Ex Taq™ II; Takara), 6.4 μl distilled H₂O, 0.8 μl forward primer, 0.8 μl reverse primer, and 2 μl of 1:10 diluted cDNA on the CFX96 Real-Time System (Bio-Rad). Each reaction was subjected to the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s in 96-well optical reaction plates (Bio-Rad). The reference gene GAPDH was used to normalize the amplification of the target genes. Each qRT-PCR analysis was performed in triplicate. Primer sequences are listed in Supplementary Table S1.

In order to identify the CaPAO expression patterns in different pepper tissues without treatment and in leaves under various stresses, qRT-PCR was performed using SYBR® Premix Ex Taq™ II (TaKaRa) in 20 μl reaction volume containing 10.0 μl SYBR® Premix Ex Taq™ II, 2.0 μl diluted cDNA, and 0.8 μl of forward and reverse primers. The amplification was completed with the following cycling parameters: 95 °C for 1 min, followed by 40 cycles of 95 °C for 10 s, 57 °C for 20 s and 72 °C for 20 s. The CaUBI3 gene (accession number: AY486137.1) was used as an internal control (reference gene) in this study (Table 1). The relative gene expression levels were calculated using the 2^−ΔΔCt comparative threshold method [39 (link)]. All samples were performed in triplicate, and each had at least three independent biological replicates.
Semi-quantitative RT-PCR was performed for analysis the expression level of CaPAO gene in transgenic tobacco, and the RD29A-F and CaPAO-R primer pair were used. The PCR cycles were 1 min at 95 °C followed by 29 cycles at 30 s at 95 °C, 30 s at 53 °C, and 60 s at 72 °C followed by an extension for 10 min at 72 °C. The PCR products were separated on 2 % agarose gels stained with ethidium bromide to detect expression degree of CaPAO gene. The NtUBI gene was used as a reference gene.