Date of Award
Doctor of Philosophy
Genetics, Development and Cell Biology
Thomas A. Peterson
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 multiple pathways for repair of DSBs induced by Ac/Ds excision and by I-SceI cutting at the same chromosomal loci. The results show that differential pathway utilization exists between the repair of Ac/Ds excision and I-SceI induced DSBs: (i) In somatic tissues, Ac/Ds induced HR preferentially utilizes SSA and/or represses GC 4 fold higher than I-SceI; (ii) In germinal tissues, repair of Ac/Ds induced DSBs favors NHEJ and strongly represses HR by 2 to 3 orders magnitude, whereas I-SceI-induced DSBs are repaired equally by NHEJ and HR; (iii) Furthermore, Ac/Ds induced germinal HR preferentially utilizes SSA and/or represses GC 5 fold higher than I-SceI; and (iv) germinal tissues preferentially utilize SSA 3 fold higher than GC compared to somatic tissues for both Ac/Ds and I-SceI induced HR. Despite these inequalities, a roughly positive correlation exists between somatic and germinal HR frequencies for both Ac/Ds and I-SceI-induced DSBs. Overall, DSB repair pathway and frequency is strongly affected by both cell type (somatic vs. germinal) and DSB agent. The striking difference of repair pathway utilization between Ac/Ds and I-SceI suggests specific role(s) of Ac/Ds in DSB repair. The hairpin intermediate generated prior to DSB formation, or Ac transposase per se, may promote NHEJ and SSA and/or repress GC. These results provide new insight into how transposons affect genome structure and also shed light on the biology of DSB-induced HR that may facilitate the development of genome modification tools for plants.
Kuang, Xianyan, "Studies of site-specific DNA double strand break repair in plants" (2010). Graduate Theses and Dissertations. 11350.