Mx3000p real time pcr system

Transcriptional changes in the MaMADSes genes responding to fruit development and ripening and 38 selected genes interacted with MaMADS24 and 49 were determined by qRT-PCR analysis on a Stratagene Mx3000 P Real-Time PCR system using SYBR® Premix Ex Taq™ (TaKaRa, Japan). The PCR amplification conditions used for all reactions were implemented as follows: 10 min at 95 °C, followed by 40 cycles of 10 s at 95 °C, 15 s at 50 °C, and 30 s at 72 °C. The relative expression levels of the target genes were calculated using the 2−ΔΔCt method^{55 (link)}. Reaction specificities for each primer pair were tested using qRT-PCR melting curve analysis, agarose gel electrophoresis and sequencing of PCR products (Supplementary Table S6). MaRPS2 (HQ853246) and MaUBQ2 (HQ853254) were used as the internal controls to normalize the expression of target genes in banana^{56 (link)}. Each treated sample contained a corresponding regularly-watered control and each sample had three independent biological replicates. The treated and control plants at each time point were sampled for expression analysis. The relative expression levels of the MaMADSes genes in each treated time point were compared with that in each time point at normal conditions.

Transcriptional changes of MebZIP genes responding to various stimuli, including osmotic, salt, cold, ABA and H₂O₂ were determined by quantitative real-time PCR analysis on Stratagene Mx3000P Real-Time PCR system using SYBR^® Premix Ex Taq™ (TaKaRa, Japan). The PCR amplification conditions used for all reactions were implemented as follows: 10 min at 95 °C, and followed by 40 cycles of 10 s at 95 °C, 15 s at 50 °C and 30 s at 72 °C. The relative expression levels of the target genes were calculated by the 2^–ΔΔCt method44 (link). Reaction specificities for each primer pairs was tested using quantitative real-time PCR melting curve analysis, agarose gel electrophoresis and sequencing PCR products (Supplementary Table S9). β-tubulin gene (TUB) and elongation factors 1α gene (EF1) were employed as internal references to normalize the transcriptional levels of target genes45 . Each treated sample contained a corresponding regularly-watered control and each sample had three independent biological replications. The treated and control plants at each time point were sampled for expression analysis. The relative expression levels of MebZIP genes in each treated time point were compared with that in each time point at normal conditions.

Transcriptional changes in MuMADS1 and MaOFP1, as well as key genes responding to fruit quality formation in transgenic lines and controls, were determined via qRT‐PCR analysis on a Stratagene Mx3000P Real‐Time PCR system with SYBR^® Premix Ex Taq™ (TaKaRa, Japan). The PCR amplification set up for every one of the reactions was: 10 min at 95 °C, then 40 cycles of 10 s at 95 °C, 15 s at 50 °C and 30 s at 72 °C. The typical expressions of the target genes were quantified by the 2^−ΔΔCT method (Livark and Schmittgen, 2001). 18S rDNA (accession number: X51576.1) was used as the internal control to normalize the expression of the target genes in tomato.

Quantitative real-time PCR was performed using SYBR Green PCR master mix (TAKARA) in a total volume of 20 μl on Mx 3000 P Real-Time PCR System as follows: 95 °C for 30 seconds, 50 cycles of 95 °C for 10 seconds, 60 °C for 30 seconds. A dissociation step was performed to generate a melting curve to confirm the specificity of the amplification. β-actin was used as the reference gene. The relative levels of gene expression were calculated by the 2^−ΔΔCt method. Primer sequences were synthesized as the Table 1.

Total RNA was isolated from the young leaf tissues (100 mg) of sugar beet using RNA-easy Isolation Reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. RNA concentrations and purity (OD_260/280) were measured with a NanoDrop 2000c, and their integrity was assessed using 1% agarose gel electrophoresis. DNase I and PrimeScript RT reagent kits (Takara, Dalian, China) were used to eliminate the genomic DNA and prepare the first-strand cDNA, respectively. Quantitative real-time reverse phase–PCR (qRT-PCR) was performed in TB Green premix Ex Taq (Takara, Dalian, China) using the Mx3000P real-time PCR system with three biological replications based on previous experiments (Pi et al. 2020 (link)). The primers for BvDREBs were designed using Primer-BLAST, and BvGAPDH was used as an internal control (Additional file 3: Table S1). The relative levels of gene expression were calculated using the 2^−ΔΔCT method.