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Plant and Cell Physiology Advance Access originally published online on January 17, 2006
Plant and Cell Physiology 2006 47(3):372-379; doi:10.1093/pcp/pcj003
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Molecular Characterization of True and Ectopic Gene Targeting Events at the Acetolactate Synthase Gene in Arabidopsis

Masaki Endo1,2, Keishi Osakabe1, Hiroaki Ichikawa1 and Seiichi Toki1,*

1 Department of Plant Biotechnology, National Institute of Agrobiological Sciences, 2-1-2, Kannondai, Tsukuba, Ibaraki, 305-8602 Japan
2 Graduate School of Life Environment Science, Tsukuba University, 1-1-1, Tennoudai, Tsukuba, Ibaraki, 305-0005 Japan

* Corresponding author: E-mail, stoki{at}affrc.go.jp; Fax, +81-29-838-8450.

Precise modification of plant genomes via gene targeting (GT) is important for the study of gene function in vivo. A reliable GT system using the protoporphyrinogen oxidase (PPO) gene in Arabidopsis was reported 4 years ago; however, there are no subsequent successful reports of GT in Arabidopsis. A previous study showed ectopic gene targeting (EGT) of the endogenous gene in two-thirds of GT plants, which was an obstacle to efficient true gene targeting (TGT). The endogenous acetolactate synthase (ALS) gene is involved in the biosynthesis of branched chain amino acids in plants and is the site of action of several herbicides. To confirm the generality of the GT system in Arabidopsis, and to characterize the EGT event in plants in detail, we converted ALS from a herbicide (imazapyr)-susceptible to a -resistant form by GT. We obtained two imazapyr-resistant plants following GT. One of the targeting events was TGT while the other was EGT. After detailed Southern blotting, PCR and nucleotide sequence analysis of the EGT plant, we determined the genomic position and structure of the ectopically targeted site. Based on our findings, we discuss the possible mechanisms of EGT in plants.

(Received September 1, 2005; Accepted January 1, 2006)
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