|
| Status |
Public on Jan 01, 2015 |
| Title |
control line 1, crl1 mutant, 4 hours after dexamethasone treatment |
| Sample type |
RNA |
| |
|
| Source name |
crl1 mutant
|
| Organism |
Oryza sativa |
| Characteristics |
tissue: stem bases of 8 day old seedlings
genotype: Taichung 65
line: 1
genotype/variation: crl1 mutant
treatment: 4 hours after dexamethasone treatment
|
| Treatment protocol |
One day after transfer of seedlings in liquid culture medium, the medium was supplemented with 10 µM dexamethasone or mock (0,5‰ v:v ethanol). Stem bases were collected 4 and 8 hours after dexamethasone treatment and immediately frozen in liquid nitrogen for RNA extraction.
|
| Growth protocol |
Dehulled and surface sterilized seeds of Oryza sativa, cv. Taichung65 were cultured in sterile closed pots containing half strength Murashige and Skoog (MS) medium and were incubated at 26°C under a 12h light/12h dark photoperiod. Seven day-old seedlings were transferred in flasks containing half strength MS liquid medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The full-length CRL1 (Os03g05510) cDNA was amplified by RT-PCR from RNA extracted from stem base and roots of Oryza sativa cv Taichung 65. CRL1-specific primers containing sequences of restriction sites adapted for the clonings. The obtained CRL1 amplicon was cloned in the SpeI/XhoI restriction sites of the binary vector pINDEX2 (Ouwerkerk et al., 2001, Planta 213, 370-378) to generate the pINDEX2-CRL1. This plasmid was transferred into Agrobacterium tumefaciens strain EHA105. Genetic transformation was performed as previously described (Sallaud et al., 2003, Theor Appl Genet 106, 1396_1408). Single locus and homozygous T2 lines were selected on the basis of the segregation of the antibiotic resistance gene carried by the T-DNA. The induction of CRL1 by dexamathasone was checked by northern blot. The capacity of transgenic lines to produce crown roots 48h after dexamethasone treatment was evaluated. On this base three independent lines expressing CRL1 after dexamethasone treatment (called DXC1, DXC2 and DXC3) were selected. One line transformed with pINDEX2 empty plasmid (called DX) was used as control.
Total RNA was extracted from frozen twenty stem bases using a RNeasy Plant Mini Kit (Quiagen) according to manufacturer’s instructions. Residual genomic DNA was removed with the RNAse-Free DNase Set (Quiagen) during RNA purification. They were quantified with a NanoQuant at 260nm wavelength and analyzed for quality on a BioAnalyzer 2100 (Agilent, CA, USA).
|
| Label |
Biotin
|
| Label protocol |
Antisense RNA strand were biotynilated according the Affymetrix (Affymetrix, CA, USA) IVT Express protocol.
|
| |
|
| Hybridization protocol |
All steps of Microarray hybridization and data processing were performed according manufacturer’s instructions (Affymetrix). Equipments were provided by Affymetrix Company. Twelve μg of biotin-labelled antisens RNAs were fragmented and hybridized to GeneChip Rice Genome Arrays (Affymetrix) for 16h at 45°C and 60rpm in a Hybridization Oven 645.
|
| Scan protocol |
Arrays were washed, labeled with phycoerythrin on the Fluidic Station 450 and read with the Scanner 3000 7G.
|
| Description |
Gene expression data from stem bases of 8 days old seedlings of crl1 mutant 4 hours after dexamethasone treatment.
|
| Data processing |
Data acquisition was done with the GeneChip Command Console. Array pictures were analyzed with MAS5 algorithm of the Expression Console software (Affymetrix).
|
| |
|
| Submission date |
Dec 12, 2013 |
| Last update date |
Jan 01, 2015 |
| Contact name |
Pascal Gantet |
| E-mail(s) |
[email protected]
|
| Organization name |
Université Montpellier 2
|
| Department |
IRD
|
| Lab |
UMR DIADE
|
| Street address |
Avenue Agropolis
|
| City |
Montpellier |
| ZIP/Postal code |
34000 |
| Country |
France |
| |
|
| Platform ID |
GPL2025 |
| Series (1) |
| GSE53272 |
Transcript profiling in stem base of crown root less 1 mutant after ectopic expression induction by dexamethasome of CRL1 |
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