Project description:Comparison between transcriptomes of dash mutants vs WT plants and ectopic expression of 35S::DASH in hairy roots versus empty vector Loss of function: 3 WT plants A17 at 8 days after pollination and 3 dash mutant plants at 8 days afer pollination. Gain of function transformants: 3 transformed plants containing empty vector and 4 transformed plants containing 35S::DASH Total RNA was extracted using a modified cetyltrimethylammonium bromide method (Verdier et al., 2008). 5ug of total RNA from each sample was purified (RNeasy MinElute Cleanup kit; Qiagen) according to the manufacturer?s instructions. RNA was quantified and evaluated for purity using an ND-1000 Nanodrop Spectrophotometer (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent). The Affymetrix M. truncatula GeneChip Array (Affymetrix) was used for expression analysis during seed development. RNA from three (for dash mutant analysis) and four (for DASH ectopic expression analysis) independent biological replicates were analysed for each time point. Probe synthesis/labelling was carried out from 500 ng of RNA using the GeneChip 3?IVT express kit, according to the manufacturer?s instructions (Affymetrix). Array hybridization, scanning, and data normalization were performed as described by Benedito et al., (2008). Each file from the hybridized Affymetrix array was exported from GeneChip Operating Software version 1.4 (Affymetrix) and imported into Robust Multiarray Average Express (Irizarry et al., 2003) for global normalization. Presence/absence call for each probe set to remove background noise was obtained using dCHIP (Li and Wong, 2001). To identify probe sets differentially expressed in dash mutant vs WT control, the R package Anapuce (J. Aubert, UMR 518 AgroParisTech/INRA) was used. For each probe set, a paired t-test was performed on the log2 expression data from three arrays (3 independent biological repeats for 8 dap data), assuming that the variance of the log expression was the same for all transcripts per genotype. Spots with extreme specific variance, too low or too high, were excluded from the statistical analysis (details on the procedure given in Gagnot et al., 2008). P-values were adjusted by the Benjamini-Hochberg method (Benjamini and Hochberg, 1995), which controls the family-wise error rate.
Project description:Hairy root lines over-expressing MtPAR using a 35S promoter compared with hairy lines over-expressing GUS gene. Hairy roots were generated in vitro using leaf explants from Medicago A17. The goal of this experiment is to prove that ectopic expression expression of MtPAR is sufficient to induce tannin biosynthesis
Project description:M. truncatula was transformed with TT2 (transparent testa2 from Arabidopsis) using Agrobacterium rhizogene and the transgenic hairy roots were used for gene profiling in comparison with empty vector control.
Project description:The hairy cell leukemia line JOK1 with low RhoH expression was stably trasfected with either an empty expression vector or this same vector expressing human RhoH. The transcriptomes of these two daughter lines were then compared by differential microarray analysis The hairy cell leukemia line JOK1 with low RhoH expression was stably trasfected with either an empty expression vector or this same vector expressing human RhoH. The transcriptomes of these two daughter lines were then compared by differential microarray analysis
Project description:Comparison of WT and efd-1 nodule transcriptome, using isolated nodules, at 4 and 10 dpi. Search of EFD possible targets by comparison of transcriptomes from empty vector-tranformed vs. 35S:EFD roots.
Project description:Transgenic tobacco hairy roots (HR) were developed by expressing the bacterial arsenite efflux pump ACR3, either at the plasma membrane or at the tonoplast of root cells. The aim of the present work was assessing the effect of the heterologous expression of Acr3 on the transcriptome of transgenic HR, in presence (0) or abscence of arsenite, in order to understand the accumulation properties of the roots developed. Our results reveal that transgenic HR expressing ACR3 at the plasma membrane displayed a completely different gene expression profile as compared to control HR (transformed with the empty vector). On the other hand, HR expressing ACR3 at the tonoplast, made through the fusion of the ACR3 protein to the tonoplast integral protein TIP1.1, showed a gene expression profile much more similar to that of control HR.
Project description:ABI3 is a B3-domain transcription factor that acts as a master regulator of seed maturation. To identify genes that are regulated by this transcription factor in the model legume Medicago truncatula, Medicago hairy roots were generated using Agrobacterium rhizogenes transformed with the genomic sequence of the ABI3 gene of Medicago. Using the Medicago NimbleGen chip, a transciptomic analysis was performed to identify differentially expressed genes compared to the GUS expressed control. Two-condition experiment, GUS versus genomic. Biological replicates: 3 control (35S::GUS), 3 treatment (35S::Genomic ABI3 sequence), independently grown and harvested. One replicate per array.
Project description:Transcriptional profiling of primary xylem tranformed cells comparing control empty vector to PtaHB-1 overexpressed Over-expression constructs were obtained by inserting the complete coding sequences of PtaHB1 and PtaHB7 from P. tremula x P. alba between the maize ubiquitin promoter (Christensen et al., 1992) and a 35S terminator into the pCambia1305.2 vector (www.cambia.org). The resulting plasmids were then transferred into A. tumefaciens strain C58 pGV2260 (Hellens et al., 2000).
Project description:Recently, AtC3H14, a CCCH-type zinc finger protein, was identified as one of the direct targets of MYB46, which is known as a master regulator of secondary wall biosynthesis. AtC3H14 and their homologs (i.e., AtC3H15 and PtrC3H14-1 from Arabidopsis and poplar, respectively) are predominantly expressed in the secondary wall forming tissues. Transgenic Arabidopsis plants overexpressing AtC3H14 (i.e., 35S::AtC3H14 plants) produced dwarfing phenotypes. 35S::AtC3H14 plants developed phloem fibers earlier than wild-type and this phenotype was more pronounced in the roots. Interestingly, ectopic secondary wall thickenings were found in both stems and roots. These phenotypic consequences are successively reproduced from the 35S::AtC3H15 and 35S::PtrC3H14-1 plants. Whole transcriptome GeneChip analysis identified that the ‘cell wall’ and ‘extracellular’-related genes are extremely over-represented in the stem tissues of 35S::AtC3H14 plants. These results suggest that AtC3H14 may act as a negative regulator of cell elongation with modification of cell wall reassembly and be involved in the secondary wall formation in Arabidopsis.