T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.
Bottom Line: Our results demonstrate that the plasmid rescue approach can be used for resolving T-DNA integration sites in Laccaria.Neither obvious sequence similarities were found between these sites and the T-DNA borders indicating non-homologous integration of the transgenes.Agrobacterium-mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.
Affiliation: Laboratorio de MicologĂa Molecular, Departamento de Ciencia y TecnologĂa, Universidad Nacional de Quilmes, Roque SĂĄenz PeĂąa 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.Show MeSH
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Mentions: We had previously shown that Agrobacterium can be used for gene transfer in L. bicolor. The TâDNA seemed to integrate mostly as a single copy and at different places in the fungal genome suggesting this method proper for nonâtargeted mutagenesis of the fungus (Kemppainen etâal., 2005). These conclusions were drown from Southern hybridization analysis knowing that it was possible that integration after all could had happened in sites sharing some common features or just in nonâcoding sequences. When Laccaria genome sequencing was completed mapping of precise integration sites became possible and AMT of the fungus could be further evaluated. In order to gain more information regarding the Agrobacterium TâDNA integration in L. bicolor we constructed the plasmid rescue binary vector pHg/pBks (Fig.â1). This plasmid does not carry homologous sequences with L. bicolor genome; its TâDNA allows the selection of fungal transgenic strains by hygromycin resistance and the rescue of the RBâgDNA junction by cutting with SacI under ampicillin selection in E. coli.
Affiliation: Laboratorio de MicologĂa Molecular, Departamento de Ciencia y TecnologĂa, Universidad Nacional de Quilmes, Roque SĂĄenz PeĂąa 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.