Quantifying Nitrogen Transporter Transcript

Based on previous studies [2 ,19 (link),24 (link),25 (link)], essential members of transporter families for NH₄⁺ (AMT1;2 and AMT1;6) and NO₃^- (NRT1;1, NRT2;4a) and genes encoding N assimilation (NR, NiR, GS1;3, GS2, Fd-GOGAT and NADH-GOGAT) were selected for the transcript analysis by quantitative RT-PCR (qPCR). Total RNA was isolated from the tissues and purified using a plant RNA extraction kit (R6827, Omega Bio-Tek, GA, USA), and trace genomic DNA was digested with DNase I (E1091, Omega Bio-Tek). Aliquots of 1 μg of total RNA were used for first-strand cDNA synthesis using the PrimeScript RT reagent kit (DRR037S, Takara, Dalian, China) in a 20-μl reaction according to the manufacturer’s instructions. PCR was performed in a 20-μl reaction including 10 μl 2× SYBR Green Premix Ex Taq II, 2 μl of cDNA and 1 μl of 20 mM primers (S1 Table) using a LightCycler 96 System (Roche). Actin2/7 was used as a reference gene [26 (link)]. Three biological replicates, each with three technical replicates, were assayed for each sample. The reference gene was included on each plate. The efficiencies of all of the PCR reactions were between 95 and 105% (S1 Table).