An Arabidopsis SHINE (SHN) gene overexpression construct was made by assembling the individual fragments (promoter, SHN gene, terminator) with appropriate compatible cohesive ends and ligated into the binary vector. The promoter comprised a CaMV 35S promoter fragment extending from -526 to the transcription start site, obtained as a 0.55 Kb HindIII-BamHI fragment from a pBS-SK+ derivative of pDH51 (Pietrzak et al., 1986) . The full length coding region of Arabidopsis SHN2 (At5g11190) was obtained as a BamHI-NotI fragment (Aharoni et al., 2004) and a CaMV 35S terminator fragment was obtained as a 0.21 Kb NotI-EcoRI fragment from a pBS-SK+ derivative of pDH51 (Pietrzak et al., 1986). The construct was made in the binary vector pMOG22 (ZENECA-MOGEN, NL), which contains a chimeric CaMV 35S-hygromycin phosphotransferase-tNos for selection during transformation. Agrobacterium-mediated transformation of Oryza sativa ssp. japonica cv. Nipponbare, plant regeneration and growth were performed (Greco et al., 2001), using the Agrobacterium strain AGL-1.
Growth protocol
Regenerated transgenic plantlets (see 'treatment protocol') were transferred to the greenhouse in hydroponic culture with a regime of 12 hours light, 28°C, 85% relative humidity and 12 hours dark, 21°C, 60% humidity.
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, two biological replicate samples of 6 pooled plants each were obtained for the transgenic rice AtSHN lines and WT. RNA was isolated using RNeasy Kit (Qiagen, USA). Following that, genomic DNA was eliminated using DNase I digestion. RNA quantity/quality measured by the Agilent 2100 Bioanalyzer (Agilent Technolgies, USA)
Label
biotin
Label protocol
RNA preparation/labeling was done using the Affymetrix 3' IVT Express kit, with biotin as label. The cRNA (or aRNA) is purified via by beads and fragmented before hybridization.
Hybridization protocol
4 µg RNA samples were hybridized to the Affymetrix Rice Genome Array. Hybridization was done using the GeneChip Hybridization, Wash, and Stain kit. The cocktail included 12.5 μg aRNA, control oligo B2, 20x hyb controls of bioB, bioC, bioD, and cre, 2X hyb mix, and DMSO. The hybe mix is heated at 99C for 5 min, 45C for 5 min, and then hybridized for 16h at 45C.
Scan protocol
Arrays were stained using the Affymetrix FS450 fluidics station with FS450_0001 fluidics protocol. Afterwards, arrays were scanned with the Affymetrix Gene Chip Scanner 3000 with HR (high resolution).
Description
Rice plants transformed with Arabidopsis SHN (AtSHN) gene
Data processing
Raw data were background corrected, normalized and summarized according to the custom CDF using RMA (Irizarry et al., 2003). The custom CDF was built by uniquely mapping 442,810 probe sequences to 35,161 rice gene-based probe sets in the following manner: (i) probes that have perfect sequence identity with a single target gene were selected, (ii) probes mapping to reverse complements of genes were annotated separately as antisense probes (not used in the above counts), and finally, (iii) probes were grouped into probe sets, each corresponding to a single gene, and probe sets having at least 3 unique sequences were retained (>98% probe sets have >=5 probes). Note that these stringent criteria used to construct the CDF make it possible to reliably measure expression values of members of multigene families (free from cross-hybridization between paralogs showing high sequence similarity).
