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    Lehrstuhl für Pharmazeutische Biologie

    Materialien/resources:

    1. High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function.

    2. In planta ORFeome analysis by large-scale over expression of GATEWAY® compatible cDNA clones: The AtTORF-Ex library

    3. GATEWAY compatible vector for plant transformation: pE-35S-HA-GW

    4. GATEWAY compatible vectors for Protoplast Two-Hybrid analysis (P2H) analysis

    5. GATEWAY compatible vectors for Bimolecular Fluorescence Complementation (BiFC)


    1. High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function.

    Genomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, molecular tools are required to characterize this ORFeome functionally. A high-throughput microtiter plate-based protoplast transactivation (PTA) system has been established that can be used in a screening approach to define which transcription factor (TF) regulates a given promoter in planta. Using to this procedure, the transactivation properties of 96 TFs can be analyzed rapidly, making use of promoter:Luciferase (LUC)-reporters and luciferase imaging. Applying GATEWAY(®) technology, we have established a platform to assay more than 700 Arabidopsis TFs. As a proof-of-principle, the ethylene response factor (ERF) family has been studied to evaluate this system. Importantly, distinct subsets of related ERF factors were found to activate specifically the well described target promoters RD29A and PDF1.2 that are under control of DRE or GCC box cis-elements, respectively. Furthermore, several applications of the PTA system have been demonstrated, such as analysis of transcriptional repressors, salt-inducible gene expression or functional interaction of signaling molecules like kinases and TFs. This novel molecular tool will improve functional studies on transcriptional regulation in plants significantly.

    For further information see Wehner et al., 2011.

    Comming soon: Protoplast transactivation facility (Possibility to test your Promotor:LUC construct for transactivation by more than 850 Arabidopsis transcription factors)

     

    2. In planta ORFeome analysis by large-scale over expression of GATEWAY® compatible cDNA clones: The AtTORF-Ex library

    Genomic approaches have generated large Arabidopsis thaliana open reading frame (ORF) collections. However, tools are required to functionally characterise this ORFeome. We have developed a batch procedure to simultaneously recombine a population of GATEWAY®-tagged full-length cDNAs into a plant expression vector (Weiste et al., 2007). A pool of Agrobacteria carrying these vectors has been used in flower-dip transformation experiments to gain a collection of transgenic lines which over express HA-tagged transcription factor (TFs) ORFs. This Arabidopsis thaliana transcription factor ORF expression (AtTORF-Ex) library can be used in various screening approaches to identify particular gene family members involved in plant development or stress response.

    For further information see Weiste et al., 2007. As a main resource, the REGIA TF ORF collection has been used.

    The collection is available under MTA.

    Currently the following seed pools of TF over expression lines are available:

    • AP2/ERF (ethylene response factor)-TF seed collection with 115 transcription factors (PDF)
    • bZIP-TF seed collection with 56 transcription factors (PDF)
    • WRKY-TF seed collection with 64 transcription factors (PDF)

         

    3. GATEWAY compatible vector for plant transformation: pE-35S-HA-GW  

    This high-copy binary vector enables plant specific expression of N-terminal HA-fusion genes under control of a CaMV 35 S promoter. The vector is suitable both for Agrobacterium and direct transformation protocols (MTA).

    pE-35S-HA-GW               Map  VNTI file  Sequence

    Selection:

    • E. coli: 100 µg/ml Ampicillin
    • Agrobacterium: 40 µg/ml Carbenicillin
    • plant cells: BASTA® 2 mM

         

    4. GATEWAY® compatible vectors for Protoplast Two-Hybrid analysis (P2H) analysis

    Based on the Arabidopsis leaf protoplast transformation protocol developed by the Sheen lab, we have set-up a simple procedure to perform two-hybrid analysis in protoplasts Ehlert et al. (2006), Weltmeier et al. (2006).

    For this approach GATEWAY® compatible vectors are available (MTA) which can be used for protein-protein-interaction analysis:

    • p35S-GAD-GW, GATEWAY® vector for GAL4 activation domain fusions               Map  VNTI file Sequence
    • p35S-GBD-GW, GATEWAY® vector for GAL4 DNA binding domain fusions           Map  VNTI file  Sequence
    • p35S-HA-GW, GATEWAY® vector for HA epitope tag fusions                              Map  VNTI file  Sequence
    • pGAL-UAS:GUS, reporter vector carrying a GAL4 BD specific cis elements (kindly provided by B. Weisshaar, Bielefeld, Germany)                                                                                       Map  VNTI file  Sequence

    Selection for all plasmids: 100 µg/ml Ampicillin

     

     5. GATEWAY® compatible vectors for Bimolecular Fluorescence Complementation (BiFC)

    BiFC or Split YFP approaches have been described by Walter et al. (2004). These GATEWAY® compatible vectors enable fusion of the YFP moieties to the N-terminus of the protein of interest. They are based on the described vectors in Walter et al. (2004) and have been applied by Weltmeier et al. (2006). These high-copy vectors are suitable for direct and Agrobacterium mediated transformation protocols (MTA).

    • pE-SPYNE-GW, N-terminus of YFP fused to ORF                                                  Map  VNTI file  Sequence
    • pE-SPYCE-GW, C-terminus of YFP fused to ORF                                                  Map  VNTI file  Sequence
    • pE-SPYNE (-) control vector                                                                             Map  VNTI file  Sequence
    • pE-SPYCE (-) control vector                                                                             Map  VNTI file  Sequence

    Selection:

    • E. coli: 100 µg/ml Ampicillin
    • Agrobacterium: 40 µg/ml Carbenicillin