Random Insertional Mutagenesis with Subtracted cDNA Fragments in Arabidopsis thaliana

  • Euna Cho (School of Biological Sciences, Seoul National University) ;
  • Kwon, Young-Myung (School of Biological Sciences, Seoul National University) ;
  • Lee, Ilha (Plant metabolosm ResearchCenter, Kyung Hee University)
  • Published : 2000.09.01

Abstract

We have evaluated a new mutagenesis strategy called random insertional mutagenesis with subtracted cDNA fragments. The cDNAs from long day Arabidopsis plants were subtracted by cDNAs from short day plants using PCR based cDNA subtraction. The subtracted cDNAs were inserted between 35S promoter and 3'-NOS terminator regardless of orientation. When the cDNA library was used for the random insertion into Arabidopsis genome by Agrobacterium-mediated transformation, approximately 15% of transformants showed abnormal development in leaf, floral organ, shoot apex. When 20 mutants were analyzed, 12 mutants showed single cDNA fragment insertion and 8 mutants showed more than 2 transgene insertions. Only two mutants among 12 mutants that have single cDNA insert showed consistent phenotype at T2 generation, suggesting the genetic instability of the mutants.

Keywords

References

  1. Transgenic Res. v.4 A heat-inducible Adh gene as a reporter for a negative selection in transgenic Arabidopsis Severin, K.;A. Wagner;F. Schoffl
  2. The 19th Annual Meeting of the Korean Society for Molecular Biology Random antisense mutagenesis in Arabidopsis for molecular genetic analysis of plant senescence Jun, J. H.;C. H. Kim;H. G. Nam
  3. Plant Physiol v.122 Activation tagging in Arabidopsis Weigel, D.;J. H. Ahn;M. A. Blazquez;J. Borevitz;S. K. Christensen;C. Fankhauser;C. Ferrediz;I. Kardailsky;E. J. Malancharuvil;M. M. Neff;J. T. Nguyen;S. Sato;Z. Wang;Y. Xia;R. A. Dixon;M. J. Harrison;C. J. Lamb;M. F. Yanofsky;J. Chory
  4. Science v.25 Activation of a plant gene by T-DNA tagging auxin-independent growth in vitro Hayashi, H.;I. Czaja;H. Lubenow;J. Schell;R. Walden
  5. Methods v.18 Gene identification by shotgun antisense Gomer, R. H.
  6. Kor. J. Genet. v.22 Inhibitory effect of antisense γ-tubulin expression on seedling growth of rice plant Jun, H. Y.;Y. K. Cha;Y. K. Kim;I. S. Han
  7. Biochem. Biophys. Acta v.1498 The DREAM-DRE interaction: key nucleotides and dominant negative mutants Ledo, F;W. A. Link;A. M. Carrion;V. Echeverria;B. Mellstrom;J. R. Naranjo
  8. Neuron v.28 Olfactory sensory axons expressing a dominant-negative semaphorin receptor enter the CNS early and overshoot their target Renzi, M.J;T.L. Wexler;J.A. Raper
  9. Science v.286 Epigenetics: regulation through repression Wolffe, A. P.;M. A. Matzke`
  10. Nucleic Acid Res. v.8 Rapid isolation of high-molecular-weight plant DNA Murray, M. G.;W. F. Thompson
  11. Biotechniques v.8 An improvement of the single-step method of RNA isolation by acid guanidinium thiocyanate phenol-chloroform extraction Puissant, C.;L. M. Houdebine
  12. Nuceic. Acid Res. v.18 Purification of mRNA directly from crude plant tissues in 15 minutes using magnetic oligo dT microspheres Jacobsen, F.S;E. Breivold;E. Hornes
  13. Cell v.76 Arabidopsis homeotic gene APETALA3 ectopic expression: transcriptional and posttranscriptional regulation determine organ identity Jack, T.;G. L. Fox;E. M. Meyerowitz
  14. Methods Mol. Biol. v.82 In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration Bechtold, N.;G. Pelletier
  15. Methods Mol. Biol. v.82 In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration Bechtold, N.;G. Pelletier
  16. Nature v.408 Analysis of the genome sequence of the flowering plant Arabidopsis thaliana The Arabidopsis Genome Initiative
  17. Plant Cell v.2 Introduction of a chimeric chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans Napoli, C.;C. Lemieux;R. Jorgensen