Rnase free dnasei

Total RNA was extracted from rice leaves using TRIzol reagent (Life Technologies, USA), and treated with RNase-free DNaseI (Amersham, USA). The quality and quantity of the purified RNA was assessed with a NanoDrop-1000 (NanoDrop, USA). First-strand cDNA was synthesized using ReverTra Ace (Toyobo, Japan). The primers for RT-PCR were as follows: 5′-CGGAAGCATCGGACAT-3′ and 5′-CCTGACGAGCGAAACG-3′ for EcTSR, 5′-GTGCTGGAGAAAGAAGG-3′ and 5′-AACGCATCAGGTGAAAT-3′ for EcGCL, 5′-CTTCATAGGAATG GAAGCTGCGGGTA-3′ and 5-CGACCACCTTGATCTTCATGCTGCTA-3′ for rice β-actin gene (Os03g0718100).

Total RNA was extracted from rice leaves using Trizol reagent (Life Technologies, USA), and treated with RNase free-DNaseI (Amersham, USA). The quality and quantity of the purified RNA was assessed with a NanoDrop-1000 (NanoDrop, USA). First-strand cDNA was synthesized using ReverTra Ace (Toyobo, Japan). Specific primer pairs were designed for the qRT-PCR of each NCA1 gene, and the specificity of these primers were evaluated using NCBI Primer-BLAST (Additional file 3 Table S2). The qRT-PCR was performed in 10 μL of reaction mixture consisting of 5 μL of 2 × SYBR Green PCR Master Mix (Toyobo, Japan), 0.2 μM of each primer, and 2 μL of appropriate diluted cDNA. Transcript levels of each gene were measured by the DNA Engine Opticon2 Real-Time PCR detection system and opticon monitor software (Bio-Rad, USA) according to the manufacturer’s instructions. The data were normalized to the amplification of the OsActin1 gene (Os03g0718100). Method of presenting quantitative real-time PCR data is the comparative C_T method (2^-ΔΔC_T method).