Rna isolater kit

The total rice RNA was extracted from the roots, stems, leaves, sheaths, and young panicles with an RNA isolater kit (Vazyme), and the first-strand cDNA was reverse transcribed using a reverse transcription kit (Vazyme). The quantitative real time RT-PCR (qRT-PCR) analysis was conducted with a real-time PCR system (qTOWER³ G, analytikjena, Jena, Germany). The Actin 1 gene was chosen as a reference gene. Three biological replicates were carried out in each experiment, and Student’s t test was used for the statistical analysis. All of the primers used in the qRT-PCR analysis are listed in Table S2 [55 (link),56 (link),57 (link),58 (link)].

Rice samples were harvested from 7 to 8 a.m. Total rice RNA from root, leaf, leaf sheath, young panicle and stem was extracted with a RNA isolater kit (Vazyme). The first-strand cDNA was reverse transcribed from total RNA (2 μg) by using a reverse transcription kit (Vazyme). The amplification was performed in a total volume of 10 μL with 0.1 μM of each primer and 1 × SYBR green PCR master mix (Vazyme) by using the CFX96 real-time PCR system (Bio-Rad). The reaction condition was as follow: 95 °C for 3 min, then 40 cycles of 95 °C for 10 s and 55 °C for 30 s. More than 3 plants were mixed for each sample. For each sample, three technical replicates on each of three biological replicates were carried out. The Actin 1 gene was chosen as an internal control. The 2^-ΔΔCT method was used to calculate relative changes in gene expression. The Student’s t test was used for statistical analysis. All qRT-PCR primer sets were listed in Additional file 16: Table S5.