Quantitative Expression Analysis of PtPHT Genes

Total RNA was isolated from roots, stems, new leaves, and old leaves using a plant RNA extraction kit (R6827, Omega Bio-Tek, GA, USA) according to the manufacturer's instructions. The first-strand cDNA was synthesized using a PrimeScript RT Reagent Kit (DRR037S, Takara, Dalian, China) following the removal of trace genomic DNA using DNase I (E1091, Omega Bio-Tek). The specific primers (Table S1) for quantitative real time PCR analysis were designed by Primer Premier 6.0 (Premier Biosoft, Palo Alto, CA, USA). We performed PCR in a 20 μl reaction mixture containing 10 μl of 2X SYBR Green Premix Ex Taq II (Bioteke, China), 2 μl of cDNA, and 1 μl of 20 mM of each primer (Table S1) using a Roche LightCycle 96 machine (Roche, Germany). PCR amplification was performed under the following conditions: one cycle of 2 min at 95°C, followed by 45 cycles at 95°C for 10 s, 55°C for 20 s, and 72°C for 20 s. Actin2/7 was used as a reference gene (Brunner et al., 2004b (link)). Three biological replicates with three technical replicates were assayed for each sample. Reactions for the reference gene were included in each plate. The relative expression levels of all the PtPHT genes were calculated using the 2^−ΔΔCT method (Livak and Schmittgen, 2001 (link)).