Journal Article

Molecular Characterization of True and Ectopic Gene Targeting Events at the Acetolactate Synthase Gene in <i>Arabidopsis</i>

Masaki Endo, Keishi Osakabe, Hiroaki Ichikawa and Seiichi Toki

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 3, pages 372-379
Published in print March 2006 | ISSN: 0032-0781
Published online March 2006 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcj003
Molecular Characterization of True and Ectopic Gene Targeting Events at the Acetolactate Synthase Gene in Arabidopsis

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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.

Keywords: Acetolactate synthase; Arabidopsis; Ectopic gene targeting; Gene targeting; True gene targeting; ALS, acetolactate synthase; BAC, bacterial artificial chromosome; DIG, digoxigenin; EGT, ectopic gene targeting; GT, gene targeting; HR, homologous recombination; IM, imazapyr; NHEJ, non-homologous end joining; PPO, protoporphyrinogen oxidase; TGT, true gene targeting; UTR, untranslated region

Journal Article.  5648 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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