Gene expression analysis
Various tissue samples of germinated seed, roots, leaf sheath, leaf
blade, node, panicle, and immature seeds were harvested from different
development stages (1-, 3-, and 16-week-old) of WT plants. To evaluate
salinity stress induced changes in gene expression, 1-week-old seedlings
were treated with 0 and 50 mM NaCl solution and root and tissue samples
were harvested at 30 min, 1 h, 6h, and 24 h after treatment. Total RNA
was extracted using TRIzol® reagent (Ambion, USA) and treated with DNAse
I (Sigma-Aldrich) according to manufacturer’s instructions. One
microgram of the total RNA was used to synthesize first strand cDNA
using random oligomers. Real-time qPCR was performed using SYBR® Green
TOP real qRT-PCR PreMIX (EnzynomicsTM, Daejeon, Korea), and the SYBR
signals were monitored using a C1000 Thermal Cycler and CFX96 Real-Time
System detection instrument (Bio-Rad Laboratories, USA). Expression
levels of the OsMTP1 and selected marker genes (Lim et al., 2014)
calculated using the 2-ΔΔCT method (Islam et al.,
2017). OsActinII was used as an internal control (Livak &
Schmittgen, 2001).