The Use Of Transposable Subsystem-directed Mutagenesis Of Arabidopsis Thaliana And Arabidopsis, Fa121 And Fa254 Gene Function

The maize transposons Activator (Ac) and Dissociation (Ds) are active in a number of plant species, including Arabidopsis. Transposons belonging to the Ac-Ds family preferentially reinsert near their original position (donor site). Several transposon systems have been described which carry markers to facilitate the identification of transposition events. Here we have used an Ac-based marked transposon system to demonstrate the feasibility of targeted gene disruption by transposons in Arabidopsis. First, we constructed a transgenic population of 365 independent lines, each harboring a T-DNA insertion(s) carrying Ds element (Ds-T-DNA). Second, we mapped 53 Ds-T-DNA insertion sites by thermal asymmetric interlaced PCR (TAIL-PCR) amplification of T-DNA flanking sequences, combined with sequence analysis of the PCR products. We show that the Ds elements in all of these Ds-T-DNA lines are capable of transposing. Finally, we examined the efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400-Kb distance from each of two Ds-T-DNA donor sites. Our results showed that these two Ds elements have similar distributions of reinsertion sites at the tested loci and the frequency of detected reinsertions even as far away as 400 Kb from the donor site is about 0.6 %. These results indicate that targeted transposon mutagenesis can be a very useful tool in reverse genetics. In order to facilitate the use of this targeted gene disruption system, we have constructed a searchable database of the mapped Ds-T-DNA insertions.