Sybr green premix pro taq hs premix

qPCRs were used to assay the copy numbers of pDL278 vector and variants in E. faecium strains (29212-pDL278, EFDO-cls, EF332-cls) to verify the stability of this vector. Plasmid levels were calculated using the 2^−ΔΔCt method. 16S rDNA was used as the housekeeping gene. The primers used are listed in Supplementary Table 1. The qPCR system (20 μl) contains the following: 10 μl of 2× SYBR green premix pro taq HS premix (Accurate Biotechnology Co., Ltd., China), 7.8 μl dd H₂O, 0.4 μl of forward and reverse primers each (10 μM), 0.4 μl of ROX reference dye (20 μM, Accurate Biotechnology Co., Ltd., China), and 1 μl template DNA (100 ng/μl). The qPCRs were performed with the following programs: denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s. Three biological replicates were performed for each sample, and each qPCR was conducted in triplicate using the ABI StepOnePlus system (Applied Biosystems Inc., Waltham, MA, United States).

For the expression analysis of BnaGYFs, total RNA of the 0, 24, 48, and 72 h postinoculation was isolated from the infected leaves using the Eastep™ Super Total RNA Extraction Kit (Promega, Madison, WI, USA). Reverse transcription was carried out using the GoScript™ Reverse Transcription System (Promega, Beijing, China). The RT-qPCR assay was carried out using 2 × SYBR Green Premix Pro Taq HS Premix (AG11702, Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China)) and a Step-One real-time fluorescence PCR instrument (Applied Biosystems, Bedford, MA, USA). The RT-qPCR reaction system contained 10 ng cDNA, 4 µM of each primer, 5 µL 2 × SYBR Green Premix Pro Taq HS Premix, 0.2 µL ROX reference dye, and 3.4 µL RNAase-free water. The RT-qPCR programming was as follows: denaturation at 95 °C for 120 s, followed by 40 amplification cycles (95 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s). BnaActin7 was used as an internal housekeeping gene. Two or more independent biological replicates and three technical replicates of each sample were performed for quantitative PCR analysis. Gene-specific primers used in the experiments are listed in Table S3.