Functional characterization of the rice root Germin-like protein gene-1 (OsRGLP1) promoter in Nicotiana tabacum

摘要

RNA extraction and cDNA synthesis: Leaves were grounded in pestle and mortar in the presence of liquid NO . Total RNA was extracted (100 mg of tissue) with RNA isolation kit (GeneJET plant RNA purification kit, Thermoscientific). RNA samples were purified from contaminated DNA after Turbo DNA-free DNase treatment. Quality and quantity was confirmed both by running on 1.5% agarose gel and using Nanodrop 2000. cDNA was synthesized using 2 µl of purified RNA per 20 µl of total reaction mixture which contained 2 µl of reaction buffer (5×), 4 µl of MgCl (25 mM), 2 µl of dNTP mix (10 mM), 0.5 µl of RiboLock RNase inhibitor (20 U), 0.6 µl of RevertAid Reverse Transcriptase (15 U) (Fermentas) and 1 µl of oligo (dT) primer (e-oligos) 25 pM (picomole) and 7.9 µl of nuclease-free water. The mixture was incubated at 42 °C for 1 h followed by 70 °C for 5 min and finally at 5 °C for 10 min. The product was stored at − 20 °C till further use.