High-frequency germinal transposition of DsALS in Arabidopsis.

Abstract

We have established an efficient transposontagging system in Arabidopsis thaliana using the Activator/Dissociation (Ac/Ds) elements from maize. This system consists of two components, a stable trans-activator, Acst, that supplies transposase, and a cis-responsive Ds element. Ds and Acst were constructed with different selectable and screenable markers to facilitate monitoring of Ds excisions and insertions as well as segregation of Ds and Acst. Fusions of the 35S, rbcS, or CHS promoters to Ac transposase were used to trans-activate DsALS, a Ds element carrying an herbicide-resistance gene. The ALS gene encoding acetolactase synthase, which confers resistance to chlorsulfuron, functioned as a versatile marker for selection of plants grown in tissue culture as well as in soil. Thirty-five Acst lines were crossed to two DsALS lines, and the resulting progeny were assayed for germinal transposition of DsALS. Trans-activation of DsALS by Acst resulted in germinal excision frequencies of up to 64% when using 35S promoter-Ac transposase fusions, up to 67% when using rbcS-transposase fusions, and up to 1% when using CHS-transposase fusions. Amongst progeny bearing terminal excisions, Southern analysis revealed that 45% from 35S-Acst crosses and 29% from rbcS-Acst crosses carried reintegrated DsALS elements. The Ac/Ds system we have developed should prove to be an effective tool for stable gene tagging in Arabidopsis.