A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre— lox

Abstract

A system for insertional mutagenesis and chromosomal rearrangement in Arabidopsis has been developed. The T‐DNA vectors are based on the maize transposon Ds, lox sites from the Cre— lox site‐specific recombination system, and transcriptional fusions expressing Ac transposase or Cre recombinase. The engineered transposon is termed Ds lox . Transposed Ds lox insertions were created by crossing plants bearing Ds lox with plants expressing Ac transposase, then simultaneously selecting for excision and re‐insertion in F 2 seedlings using the herbicides chlorsulfuron and phosphonothricin, respectively. F 2 plants bearing stable Ds lox insertions were identified by scoring for the absence of the Ac transposase T‐DNA, using a novel, visual marker in that T‐DNA. Two independent Ds lox insertions were characterized and placed 5.6 and 16.5 cM from their T‐DNA lox , which mapped close to m506 on chromosome 4. Plants bearing either of the two different transposed Ds loxs and T‐DNA lox were crossed to plants expressing Cre recombinase, which catalyzed recombination between the lox site in transposed Ds lox and the lox site in T‐DNA lox. Lox—lox recombinants were identified selectively amongst progeny of these crosses. Molecular and genetic analysis of the lox—lox rearrangements indicated that both were inversions. The smaller inversion was germinally transmitted from generation to generation as a simple trait, whereas the larger inversion was not transmitted to progeny of plants bearing the rearrangement.