Gene expression analysis by qRT-PCR
For gene expression analysis,
seedlings of same growth status were
treated with 0.5 mM KNO3 + 50 µM Fe, 15 mM
KNO3 + 50 µM Fe, 0.5 mM KNO3 -Fe (-Fe
solution supplemented with 200 µM ferrozine) or 15 mM
KNO3 -Fe (-Fe solution supplemented with 200 µM
ferrozine) solutions for 1, 3, 5 and 7 days, respectively. Roots were
collected, washed and frozen in liquid nitrogen. The roots were grinded
into powder with a mortar and pestle. Total RNA was extracted with an
Omini Plant RNA Kit (CWBIO, Beijing, China) following the manufacturer’s
protocol. cDNA was prepared from 1 µg total RNA using the
PrimeScriptTM RT reagent Kit (Takara, Dalian, China).
An Ultra SYBR Mixture (CWBIO, Beijing, China) was used to detect the
expression levels of genes using qRT-PCR (ABI, StepOnePlus, USA).MdActin (GenBank accession No.:CN938023) was selected as an
internal control gene. Results were based on the average of three
replicate experiments. All primers used in this study was shown in Table
1.