|
| Status |
Public on Nov 20, 2015 |
| Title |
OsMADS26 over-expressing line 2, osmotic stresscondition, rep1 |
| Sample type |
RNA |
| |
|
| Source name |
over-expressing line transformed with a PC5300.OE plasmid containing the OsMADS26 cDNA cultivated in in osmotic stress condition (100 mM manitol)
|
| Organism |
Oryza sativa |
| Characteristics |
tissue: root and shoot
age: 7 day old seedlings
cultivar: Nipponbare
|
| Treatment protocol |
The isolation of OsMADS26 (Os08g02070) cDNA from O. sativa cv Nipponbare was achieved by RT-PCR. For over-expression of OsMADS26, a BP tailed OsMADS26 PCR-amplified cDNA was cloned with the BP recombinase in a modified pCAMBIA 1300 binary vector for over-expression named PC5300.OE where the Ccdb gene surrounded by the BP recombination sites were cloned between the constitutive promoter of ubiquitin gene from maize and the terminator of the nopaline syntase gene from Agrobacterium. tumefaciens (J.C. Breitler, CIRAD, unpublished). The plasmid named PC5300.OE-PC8 was transferred into A. tumefaciens strain EHA105. For down-regulation of OsMADS26 by RNA interference, the BP tailed amplified GST1 (targeting the 5’UTR of OsMADS26 mRNA) or GST2 (targeting the 3’UTR of OsMADS26 mRNA) were cloned by BP recombination in the pDON207 entry plasmid (Invitrogen) and transferred with the LR recombinase (Invitrogen) in the siRNA binary plasmid pANDA (Miki and Shimamoto, 2004, Plant Cell Physiol 45, 490_495). The resulting plasmids, were mobilized into A. tumefaciens strain EHA105 for plant transformation. Transgenic plants were obtained by co-culture of seed embryo-derived callus with A. tumefaciens strain EHA105 carrying the adequate binary plasmids following the procedure detailed in (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 and Southern blot analysis. The over-expression or down-regulation of OsMADS26 in selected transgenic lines was assessed by RT-qPCR using specific primers. On this base two independent lines ove-rexpressing OsMADS26 (called OX1 and OX2) or one line down-regulated for OsMADS26 obtained by the expression of each of the two GST designed to target OsMAS26 5’UTR or 3’UTR (called DR5 or DR3 respectively) were selected for transcriptome analysis. One transgenic lines obtained by transformation with each PC5300.OE or pANDA empty plasmids (called OX0 or DR0 respectively) were used as control.
Osmotic stress was obtained by adding Manitol (100 mM) to the culture medium.
|
| Growth protocol |
dehulled and surface sterilized seeds of Oryza sativa, cv. Nipponbare were incubated in sterile distilled water in a growth chamber (16 h of light per day, 500 µE m-2 s-1, 28°C/25°C day/night) for 2 days at 25°C. Imbibed seeds were transferred in square Petri dishes (245 mm x 245 mm, Corning, 7 seeds per dish) containing 250 ml of half strength Murashige and Skoog (Duchefa) standard medium (MS/2) solidified with 8 g L-1 of agarose type II (Sigma). These dishes were transferred and placed vertically in a growth chamber at 28°C under 16h light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Roots and shoots of 7 day-old seedlings of 7 plantlets were collected, pooled and used for transcriptome analyses.total RNA was extracted from 100 mg of frozen leaves and roots 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. Two independent biological experiments were used for each studied plant line.
|
| 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 roots and shoots of 7 days old transgenic seedlings over-expressing OsMADS26 and cultivated in osmotic stress (100 mM manitol) condition
OX2_M_1
|
| Data processing |
Data acquisition was done with the GeneChip Command Console. Array pictures were analyzed with MAS5 algorithm of the Expression Console software (Affymetrix).
Default parameters were applied, global scaling method was used to normalize data (TGT value set at 100).
|
| |
|
| Submission date |
Feb 03, 2014 |
| Last update date |
Nov 21, 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) |
| GSE52640 |
Transcript profiling of transgenic rice lines where the OsMADS26 gene is over-expressed or down growing cultivated in standard or osmotic stress condition |
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