Hiscript q rt supermix kit

Total RNA was extracted using RNA isolator reagent (Vazyme, Nanjing, China) and reverse transcribed into cDNA using a HiScript QRT SuperMix kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Quantitative PCR (qPCR) reactions contained 10 μL 2× AceQ qPCR SYBR green master mix (Vazyme), 0.1 μg cDNA, and 7.5 pmol of each of the gene-specific primers (Table S2) in a final volume of 20 μL and was run in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). FonActin was used as an internal control to normalize the data, and the relative expression of the genes tested was calculated using the 2^–ΔΔCT method.

Total RNA was extracted from 100 mg of tomato root or leaf tissue using a total RNA kit (Omega Bio-tek, Inc., Georgia, USA) in accordance with the manufacturer’s instructions (genomic DNA was removed). A sample of 1 μg total RNA was reverse-transcribed to synthesize cDNA using a HiScript QRT SuperMix Kit (Vazyme Co., Nanjing, China). qRT-PCR was performed using SYBR Green PCR Master Mix (Vazyme Co.) on a StepOnePlus Real-time PCR Detection System (Applied Biosystems). The specific primers used for qRT-PCR are listed in Supplementary Table S3. The PCR protocol was as follows: denaturation at 95 °C for 5 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 58 °C for 30 s, and extension at 72 °C for 10 s. At the end of each PCR cycle, a dissociation curve was generated using software provided with the StepOnePlus Real-time PCR Detection System to verify that a single product was amplified. Three biological and three technical replicates were used to determine the mRNA expression level of the target gene, and the generated threshold cycle (C_T) was used to calculate transcript abundance relative to that of the housekeeping gene Actin (Mascia et al., 2010 (link)). The mRNA quantification procedure was based on the method of Livak and Schmittgen (2001) (link).

Total RNA was extracted from ‘White heaven’ leaves using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. After DNase I (Takara, Bio, Beijing, China) treatment, 1 μg of RNA was subject to a reverse transcription reaction using the HiScript Q RT SuperMix Kit (Vazyme, Nanjing, China). The degenerate primers were designed (Supplementary Table S1) and the partial conserved sequence of LlHsfA4 was amplified by homology-based cloning.The full-length sequence of LlHsfA4 was then obtained by 5′- and 3′- one step Full Race Kit (Takara, Japan). The conserved domain prediction was carried out using the DNAMAN software and NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 28 August 2021) website. Thephylogenetic tree was analyzed using the ClustalW 2.0 and MEGA 5.0 software.