Rabbit Anti-Human LIG4 (Phospho-Thr650) polyclonal antibody

Evidence for the involvement of the nonhomologous end joining (NHEJ) pathway in Agrobacterium-mediated transferred DNA (T-DNA) integration into the genome of the model plant Arabidopsis remains inconclusive. Having established a rapid and highly efficient Agrobacterium-mediated transformation system in rice (Oryza sativa) using scutellum-derived calli, we examined here the involvement of the NHEJ pathway in Agrobacterium-mediated stable transformation in rice. Rice calli from OsKu70, OsKu80 and OsLig4 knockdown (KD) plants were infected with Agrobacterium harboring a sensitive emerald luciferase (LUC) reporter construct to evaluate stable expression and a green fluorescent protein (GFP) construct to monitor transient expression of T-DNA. Transient expression was not suppressed, but stable expression was reduced significantly, in KD plants. Furthermore, KD-Ku70 and KD-Lig4 calli exhibited an increase in the frequency of homologous recombination (HR) compared with control calli. In addition, suppression of OsKu70, OsKu80 and OsLig4 induced the expression of HR-related genes on treatment with DNA-damaging agents. Our findings suggest strongly that NHEJ is involved in Agrobacterium-mediated stable transformation in rice, and that there is a competitive and complementary relationship between the NHEJ and HR pathways for DNA double-strand break repair in rice.

Gene targeting is a technique of introducing a genetic trait at a predetermined site within a genome; it is also used to eliminate undesirable chromosomal regions from the relevant genome. Thus far, replacement-type recombination between two homologous regions separated by a large nonhomologous sequence has been hardly achieved probably due to the low frequency of homologous recombination in filamentous fungi. In this study, we report the successful and highly efficient deletion by replacement-type recombination of up to 470-kb regions of chromosome 8 and 200-kb region in chromosome 3, which includes a homologue of aflatoxin gene cluster, by nonhomologous end-joining deficient strains of Aspergillus oryzae. Our study results indicate that the deficiency of nonhomologous end-joining increases the distance of nonhomologous regions in replacement-type recombination, i.e., the possible deletion range in generation of large chromosomal deletion by one cycle of replacement-type recombination is increased in nonhomologous end-joining deficient strains. (C) 2009 Elsevier Inc. All rights reserved.