Sybr green dye method

RNA was isolated from the leaves using a TRIzol kit (Accurate Biotechnology, Changsha, China). cDNA was synthesized using a reverse transcription kit (Accurate Biotechnology, Changsha, China) and real-time PCR was performed using the SYBR Green dye method (Accurate Biotechnology, Changsha, China). The PCR primers were designed using Primer Premier 5 software (Vancouver, BC, Canada) and are listed in Table S8. The qPCR reaction was accomplished in a Bio-Rad CFX system, with a 10 µL reaction system containing 5 µL of TB Green premix Ex Taq II, 1 µL of cDNA, 0.5 µL each of forward and reverse primers, and 3 µL of RNase-free water. The qPCR reaction conditions were set according to the manufacturer’s instructions. SiActin was used as an internal standard, and the expression levels of each gene were calculated using the 2^−ΔΔCt method.

The RNA was isolated from leaves using a TRIzol kit (Accurate Biotechnology, Changsha, China). The quality of RNA samples was verified by agarose gel electrophoresis (Figure S6). Then, the cDNA was synthesized using a reverse transcription kit (Accurate Biotechnology, Changsha, China), and real-time PCR was carried out using the SYBR Green dye method (Accurate Biotechnology, Changsha, China). The PCR primers were designed using Primer Premier 5 software (Vancouver, BC, Canada), and the primers are listed in Table S7. The RT-PCR reaction was accomplished in a Bio-Rad CFX system, with a 20 µL reaction system containing 10 µL of 2× SYBR Green Pro Taq HS premix, 2 µL of cDNA, 0.4 µL of each of forward primer and reverse primer, and 7.2 µL of RNase free water. The reaction conditions of RT-PCR were according to the instructions. The melting curve analysis of primers used for RT-PCR is presented in Figure S7. SiActin (SETIT_026509mg) was used as an internal standard, and the expression levels of each gene were calculated by the 2^−∆∆Ct method [56 (link)]. The heat map of SiCYP450 expression was constructed by TBtools, the expression value was standardized by log₂. Three biological replicates were conducted for the transcript profiles of genes. Each independent experiment was repeated at least three times.

The acquisition of RNA from leaves was accomplished with TRIzol kit (Accurate Biology, Changsha, China), cDNA was synthesized using reverse transcription kit (Accurate Biology, Changsha, China), and real-time PCR was performed with SYBR Green dye method (Accurate Biology, Changsha, China). PCR primers were designed using Primer Premier 5 software (Table S6). The qRT-PCR reaction was performed in a Bio-Rad CFX system, a 20 µL reaction system containing 10 µL 2× SYBR Green Pro Taq HS premix, 2 µL cDNA, 0.4 µL each of forward primer and reverse primer, 7.2 µL RNase free water. The following cycling conditions were used: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The SiActin (SETIT_026509mg) was used as internal standard, and the expression levels of each gene were calculated by the 2^−ΔΔCt method. The heat map of SiGSTs expression condition was constructed by TBtools, expression value was standardized by Log2. Each independent experiment was repeated at least three times.