Expression analysis of response to external status of ammonium in rice roots.

Nitrogen is major nutrient for plant growth. Two forms for inorganic nitrogen are available for plant, ammonium and nitrate. External status of them affects largely plant growth and production. Generally, root is a sole organ to uptake nitrogen. Hence, identifying regulatory genes depend on nitrogen status in roots is important to improve sustainable production or rice. To isolate the candidate gene, array experiments were performed. Consequently, we isolated genes which showed marked accumulation in deficient and sufficient concentrations of ammonium in roots. Rice Oligo DNA Microrray 4X44K RAP-DB (Agilent Technologies) was used in this research. Total RNA was extracted from roots of Koshihikari grown for 10 days in 0.005 and 1 mM NH4Cl with RNeasy Plant Kit (QIAGEN) in accordance with recommend protocol. We performed four biological replicate for both treatments. Before array analyses, RNA integrity was confirmed with Agilent 2100 Bioanalyzer. Array analysis was performed in accordance with recommend protocol as follow. Total RNA (100ng) was labeled with Cy3 using Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies). Cy-labeled cRNA (0.00165 mg) was fragmented for 30 min at 60 degree and the hybridized for 17 hrs at 65 degree with gentle shaking (10 rpm) using Gene Expression Hybridization Kit (Agilent Technologies). Following, the arrays were washed using Gene Expression Wash Buffers Pack (Agilent Technologies) and then scanned using a scanner, Agilent DNA Microarray Scanner (G2565CA). For data processing, Agilent Feature Extraction software was used for delineation and measurement of the signal, normalization of intensity, and measurement of local background at individual spot in accordance with the manufacturer’s instructions.