Studies of site-specific DNA double strand break repair in plants
type: CreativeWork
description: The maize transposon Ac/Ds transposes by a “cut and paste"” mechanism, leaving a site-specific DSB at the Ds locus for repair. Previous studies in maize and Arabidopsis showed that Ac/Ds excision can stimulate homologous recombination between tandem duplicated repeats and between ectopic homologies. In this study, we tested the efficacy of such Ac/Ds excision-induced homologous recombination to achieve gene targeting in Arabidopsis. A defective visual-selective dual marker harboring Ds element is transformed into Arabidopsis as target locus, and donor homology is provided by Agrobacterium-mediated T-DNA by floral dipping. The DSB inducer Ac is either first crossed into target lines or co-transformed with donor T-DNA, and several independent germinal recombinants have been recovered from both strategies with gene targeting frequency of 0.3-2.0X10(-3). We also attempted gene targeting by ectopic recombination, with donor homology provided by the concomitant integrated T-DNA copies in genome. With this approach the gene targeting frequency turns out less than 6.7X10(-6). Our results suggest that Ac/Ds transposon-induced homologous recombination may provide an alternative gene targeting strategy. The implications of these results for plant gene targeting are discussed. Multiple pathways are used to repair Double Strand Breaks (DSBs), including Nonhomologous End Joining (NHEJ) and Homologous Recombination (HR) which can take Single Strand Annealing (SSA) or Gene Conversion (GC) pathway. DSB repair in plants has been extensively studied using site-specific DSB agents, including two mechanistically different inducers transposon Ac/Ds and endonuclease I-SceI. A direct comparison between Ac/Ds and I-SceI in DSB repair, however, is lacking due to the diversity of systems used among previous studies. In addition, only a few DSB repair studies addressed germinal recombination frequencies. In this study, we developed three constructs (HRS1, 2 and 3) that allow comparison of mult…
abstract: The maize transposon Ac/Ds transposes by a “cut and paste"” mechanism, leaving a site-specific DSB at the Ds locus for repair. Previous studies in maize and Arabidopsis showed that Ac/Ds excision can stimulate homologous recombination between tandem duplicated repeats and between ectopic homologies. In this study, we tested the efficacy of such Ac/Ds excision-induced homologous recombination to achieve gene targeting in Arabidopsis. A defective visual-selective dual marker harboring Ds element is transformed into Arabidopsis as target locus, and donor homology is provided by Agrobacterium-mediated T-DNA by floral dipping. The DSB inducer Ac is either first crossed into target lines or co-transformed with donor T-DNA, and several independent germinal recombinants have been recovered from both strategies with gene targeting frequency of 0.3-2.0X10(-3). We also attempted gene targeting by ectopic recombination, with donor homology provided by the concomitant integrated T-DNA copies in genome. With this approach the gene targeting frequency turns out less than 6.7X10(-6). Our results suggest that Ac/Ds transposon-induced homologous recombination may provide an alternative gene targeting strategy. The implications of these results for plant gene targeting are discussed. Multiple pathways are used to repair Double Strand Breaks (DSBs), including Nonhomologous End Joining (NHEJ) and Homologous Recombination (HR) which can take Single Strand Annealing (SSA) or Gene Conversion (GC) pathway. DSB repair in plants has been extensively studied using site-specific DSB agents, including two mechanistically different inducers transposon Ac/Ds and endonuclease I-SceI. A direct comparison between Ac/Ds and I-SceI in DSB repair, however, is lacking due to the diversity of systems used among previous studies. In addition, only a few DSB repair studies addressed germinal recombination frequencies. In this study, we developed three constructs (HRS1, 2 and 3) that allow comparison of mult…
author: Kuang, Xianyan
language: en
date published: 2010-01-01
date modified: 2026-07-24T02:39:19Z
provider: Iowa State University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Iowa State University

PropertyValue
property ID: DOI
value: 10.31274/etd-180810-2459