Realmastermix kit

To investigate whether ethylene biosynthesis and signaling were involved in the plant response to Cd stress, the relative expression of genes encoding for ethylene biosynthesis, and of perception and signaling proteins were measured in seedlings that were treated with different concentrations of CdCl₂ for 4 days. Total RNA was isolated from 100 seedling roots using TRIzol solution (Invitrogen, Carlsbad, CA, USA). Two micrograms of total RNA was used for first-strand cDNA synthesis using RevertAid Reverse Transcriptase and Oligo d(T)primers (Takara, Dalian, China). The cDNA yield was measured according to the PCR signal generated from the internal standard, with the housekeeping gene Actin 2 used as the internal control. The primers that were used in the quantitative RT-PCR are listed in Table 1 (Liu et al., 2010 (link); Lin et al., 2013b (link); Li et al., 2014 (link)).
Quantitative RT-PCR was performed using a RealMasterMix kit (Tiangen, Beijing, China) with 25 cycles as follows: 94°C for 30 s, 58°C for 30 s and 72°C for 30 s, followed by 72°C for 10 min gene expression quantifications was performed using the relative 2^-ΔΔCt method (Livak and Schmittgen, 2001 (link)). All experiments were performed in triplicate for each treatment.

RNA was isolated from whole bodies of 3-day-old male and female flies using Trizol Reagent (TIANGEN, Beijing) according to the manufacturer’s protocol. RNA was reverse transcribed using Fast cDNA Reverse Transcription Kit (TIANGEN, Beijing). The Quantitative real-time PCR assay was performed using Applied Biosystem Step One Real Time PCR system (Applied Biosystem, Foster, CA, USA), RealMasterMix kit and SuperReal PreMix Plus kit (TIANGEN, Beijing). The sequences of primers are shown in Supplementary S1 Table.

For quantitative reverse transcription–PCR (qRT–PCR) analysis, whole seedlings or root tip tissues were harvested and frozen in liquid nitrogen for RNA extraction. RNA was extracted using the RNeasy kit (Qiagen). First-strand cDNA was synthesized from 2 μg of total RNA using Superscript III reverse transcriptase (Invitrogen) and oligo(dT) primers. qRT–PCRs were performed using a cycler apparatus (Bio-Rad) and the RealMasterMix kit (SYBR Green, Tiangen) according to the manufacturer’s instructions. The expression levels of the target genes were normalized to those of ACTIN2. Statistical significance was evaluated by Student’s t-test. The primers used to quantify gene expression levels are listed in Supplementary Table S1 at JXB online.

Plasmids and genomic DNA were extracted using the Plasmids mini kit and DNA purification kit (Tiangen, Beijing, China), respectively. PrimerSTAR HS DNA polymerase (Takara, Dalian, China) was used for PCR. Restriction endonucleases and T4 DNA ligase were purchased from Thermo Scientific (Waltham, USA). Total RNA was extracted using RNA extraction kit (Bio Flux, Beijing, China). The RNA was reversely transcribed into cDNA using the Revert Aid™ First Strand cDNA synthesis kit (Fermentas, Shanghai, China). RT-PCR was performed using the Real Master Mix kit (Tiangen, Beijing, China). The intracellular concentration of NADPH/NADP⁺ was determined by using NADPH/NADP⁺ Kit (Beyotime Biotechnology, Shanghai, China).

The relative qPCR was performed to calculate the expression levels of N463r-C7 and its seven mutated proteins in helix XIII (TM XIII). Strains of KNabc/D1, D2, N463r-C7 and its mutants were cultured in LBK medium at 37°C to late exponential phase. Then, cells were harvested and used for RNA extraction, by using EasyPure RNA kit (Transgen). The cDNA was synthesized by the HiScript Q RT SuperMix for Qpcr (Vazyme Biotech) and used as the templates for qPCR analysis. Quantitative PCR was carried out in a MyiQ2^TM iCycler (two-color real-time PCR detection system) (Bio-Rad) with a Real Master mix kit (SYBR Green, Tiangen) according to the manufacturer’s instructions. The primers used in this procedure are listed in Supplementary Table S2 and 16S rRNA was used as an internal reference for each mutant. Three biological replicates were used for qPCR and at least four repeats for each sample, after which the average threshold cycle (Ct) was calculated for each sample. By using the Ct values of nhaD2 as a baseline, the relative fold changes in gene expression were calculated by 2^-ΔΔCt method. Statistical analysis was performed on 2^-ΔΔCt values using a paired Student’s t-test.