Plant & Animal Genome V Conference
Town & Country Hotel, San Diego, CA, January 12-16, 1997.
PAG-V: W17 - TARGETED INSERTION MUTAGENESIS WITH THE EN-I TRANSPOSON SYSTEM IN ARABIDOPSIS
W17
TARGETED INSERTION MUTAGENESIS WITH THE EN-I TRANSPOSON SYSTEM IN ARABIDOPSIS
PEREIRA, ANDY, Mark G.M. Aarts, Elly Speulman, Peter L.J. Metz, Willem J. Stiekema
Centre for Plant Breeding and Reproduction Research (CPRO-, DLO), PO Box 16, 6700AA Wageningen, The Netherlands
The maize Enhancer-Inhibitor (En-I or Spm-dSpm) transposable element system introduced into Arabidopsis displays continuous and frequent transposition. The transposon system consists of an En-transposase source under control of the CaMV35S promoter, which mediates `constitutive' transposition of the mobile I-elements throughout plant development and often leads to transposon multiplication. About 20 tagged mutants and 5 cloned genes have been isolated by a random tagging strategy. Targeted tagging with linked I elements was shown to work in the recovery of tagged Cer6 and Clv1 mutants. The numerous tagged mutants together with a resource of about 30 mapped I elements, now facilitate targeted tagging with linked transposons of specific genes with a distinguishable mutant phenotype. To produce a `mutation machine' in Arabidopsis and recover inserts in any gene of interest, this linked transposition behavior was utilized to produce plant populations saturated for independent transpositions in specific chromosomal regions. Plant populations, with an I element transposing on chromosome 4, were organized in pools and the extracted DNA used for PCR screening with primers derived from sequenced genes in chromosome 4. Many inserts were found in genes identified by the ESSA chromosome 4 sequenced region. Target genes at different distances from the mapped I element, are being screened to estimate mutagenesis frequencies. For a `targeted gene inactivation' strategy of the whole genome, populations containing multiple (20-40 per plant) independent I element insertions, are produced which can be used for PCR screening of inserts in any gene.