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Plant and Cell Physiology Advance Access published online on January 17, 2006
Plant and Cell Physiology, doi:10.1093/pcp/pcj003
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Plant and Cell Physiology 2006 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received September 1, 2005
Accepted January 1, 2006

Regular Paper

Molecular Characterization of True and Ectopic Gene Targeting Events at Acetolactate Synthase Gene in Arabidopsis

Masaki Endo 1, Keishi Osakabe 2, Hiroaki Ichikawa 2, and Seiichi Toki 2 *

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

* To whom correspondence should be addressed.
Seiichi Toki, E-mail: stoki{at}affrc.go.jp


  Abstract

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 four 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.

Keywords: Gene targeting; true gene targeting; ectopic gene targeting; acetolactate synthase; Arabidopsis.
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