Versatile Gene-Specific Sequence Tags for Arabidopsis Functional Genomics: Transcript Profiling and Reverse Genetics Applications

  1. Pierre Hilson1,2,17,
  2. Joke Allemeersch3,
  3. Thomas Altmann4,
  4. Sébastien Aubourg1,2,
  5. Alexandra Avon2,
  6. Jim Beynon5,
  7. Rishikesh P. Bhalerao6,
  8. Frédérique Bitton2,
  9. Michel Caboche2,
  10. Bernard Cannoot1,
  11. Vasil Chardakov7,
  12. Cécile Cognet-Holliger8,
  13. Vincent Colot2,
  14. Mark Crowe9,
  15. Caroline Darimont10,
  16. Steffen Durinck3,
  17. Holger Eickhoff11,16,
  18. Andéol Falcon de Longevialle2,
  19. Edward E. Farmer10,
  20. Murray Grant7,
  21. Martin T.R. Kuiper1,
  22. Hans Lehrach11,
  23. Céline Léon2,
  24. Antonio Leyva12,
  25. Joakim Lundeberg13,
  26. Claire Lurin2,
  27. Yves Moreau3,
  28. Wilfried Nietfeld11,
  29. Javier Paz-Ares12,
  30. Philippe Reymond10,
  31. Pierre Rouzé1,
  32. Goran Sandberg6,
  33. Maria Dolores Segura12,
  34. Carine Serizet1,2,
  35. Alexandra Tabrett5,
  36. Ludivine Taconnat2,
  37. Vincent Thareau1,2,
  38. Paul Van Hummelen14,
  39. Steven Vercruysse1,
  40. Marnik Vuylsteke1,
  41. Magdalena Weingartner4,
  42. Peter J. Weisbeek15,
  43. Valtteri Wirta13,
  44. Floyd R.A. Wittink15,
  45. Marc Zabeau1, and
  46. Ian Small2,17
  1. 1 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, B-9052 Gent, Belgium
  2. 2 Unité de Recherche en Génomique Végétale (INRA-CNRS-UEVE), F-91057 Evry CEDEX, France
  3. 3 Department Electrical Engineering (ESAT), Faculty of Engineering, Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium
  4. 4 Universität Potsdam, Institut für Biochemie und Biologie,- Genetik-, c/o Max-Planck-Institut für molekulare Pflanzenphysiologie, D-14476 Golm, Germany
  5. 5 Horticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom
  6. 6 Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, S-901 83, Umeå, Sweden
  7. 7 Department of Agricultural Sciences, Imperial College London, Wye, Ashford, Kent TN25 5AH, United Kingdom
  8. 8 Station de Génétique et Amélioration des Plantes, INRA, F-78026, Versailles CEDEX, France
  9. 9 John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom
  10. 10 Gene Expression Laboratory, Department of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne, Switzerland
  11. 11 Max-Planck-Institute for Molecular Genetics, Department of Vertebrate Genomics, D-14195 Berlin-Dahlem, Germany
  12. 12 Department of Plant Molecular Genetics, Centro Nacional de Biotecnología-CSIC, E-28049 Madrid, Spain
  13. 13 Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology (KTH), S-106 91 Stockholm, Sweden
  14. 14 VIB MicroArray Facility, UZ Gasthuisberg, B-3000 Leuven, Belgium
  15. 15 Department of Molecular Genetics, University Utrecht, NL-3584 CH Utrecht, The Netherlands

Abstract

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.

Footnotes

  • Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2544504.

  • [Supplemental material is available online at www.genome.org. The pAgrikola sequence data have been submitted to GenBank under accession no. (AY568055) and the transcript profiling data to Array Express under accession nos. (E-MEXP-30 and E-MEXP-65). The GST amplicons and CATMA spotted microarrays are available from the Nottingham Arabidopsis Stock Centre (NASC; http://nasc.nott.ac.uk). NASC will also distribute the cloned GST repertoires. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: C. Helliwell, M. Salanoubat, and P. Waterhouse.]

  • ↵17 Corresponding authors. E-MAIL pierre.hilson{at}psb.ugent.be; FAX 32 9 331 38 09. E-MAIL small{at}evry.inra.fr; FAX 33 1 60874510.

  • ↵16 Present address: Scienion AG, D-12489 Berlin, Germany.

    • Accepted May 12, 2004.
    • Received March 5, 2004.
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  1. doi: 10.1101/gr.2544504 Genome Res. 14: 2176-2189 Cold Spring Harbor Laboratory Press

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