varirty: a Japonica sub-species of Oryza sativa "Wuyunjing #7". growth stage: 23 days after planting. tissue: roots
Extracted molecule
total RNA
Label
Cy5
Description
Plant material and growth condition: The cultivar of rice used in the experiments was a Japonica sub-species of Oryza sativa "Wuyunjing #7". Seeds were surface sterilized, placed individually in the hole of a nylon-net which was submerged into water for germination in a growth chamber for 10 days. The plantlets were then transplanted onto a hydroponics system in a temperature and light controlled glasshouse. During the growing period, the temperature ranged between 22 to 24 0C at night and 30 to 32 0C at day-time, and photo-period was maintained 14h/10h (light/dark).
Nutritional treatments and plant sampling: The transplanted plantlets were first supplied with full nutrition solution containing 1.25 mM ammonium nitrate as N source for 12 days, followed by a solution with N completely removed for one week. After that, the plants were re-supplied with four different treatments: ammonium nitrate (1:1 ratio), only ammonium, only nitrate and continued N starvation for further 96 h with twelve replicates for each of the treatments. The full nutrition concentration formula of the major elements is described in Table 1. In addition, the solution contained 2-mM Na2SiO4 and integrated mixture of micro-nutrients (in µM): 20.0 FeSO4, 36.7 H3BO4, 9.1 MnCl2, 0.77 ZnSO4, 0.32 CuSO4, and 0.39 H2MoO4. pH of the solution was adjusted to 5.8. The solution was replaced completely once every two days before the treatments and everyday during the treatments. After weighting, both the fresh roots and leaves of each treatment were sampled at 96-h after the treatment initiation.
RNA preparation, array hybridization and data analysis: We used an RNeasy Plant Mini Kit (Qiagen, Shanghai, China) to extract total RNA. RNA concentration and quality were analyzed by NanoDrop (NanoDrop Technologies) and 2100 Bioanalyzer (Agilent Technologies). We used Agilent low RNA input fluorescent linear amplification kit for fluorescent cDNA synthesis, fluorescent cRNA amplification and synthesis. The hybridization and washing were followed Agilent 60-mer oligo microarray processing protocol. The data scanning, quantification, and processing were done as described by Yazaki et al. (2004).
Yazaki, J., Shimatani, Z., Hashimoto, A., Nagata, Y., Fujii, F., Kojima, K., Suzuki, K., Taya, T., Tonouchi, M., Nelson, C., Nakagawa, A., Otomo, Y., Murakami, K., Matsubara, K., Kawai, J., Carninci, P., Hayashizaki, Y., and Kikuchi, S. (2004). Transcriptional profiling of genes responsive to abscisic acid and gibberellin in rice: phenotyping and comparative analysis between rice and Arabidopsis. Physiol. Genomics 17, 87-100.
