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T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

Kemppainen M, Duplessis S, Martin F, Pardo AG - Microb Biotechnol (2008)

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

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Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λBstE II, L. bicolor wild type (Wt) and transgenic strains.
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f2: Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λBstE II, L. bicolor wild type (Wt) and transgenic strains.

Mentions: Laccaria bicolor dikaryotic strain S238N (heterokaryotic, the phase in the fungal life cycle which forms symbiosis with tree roots) was transformed with Agrobacterium tumefaciens AGL‐1 carrying the pHg/pBks vector. The transformation efficiency with the given plasmid was 75% calculated from the appeared hygromycin resistant growing points against the total number of fungal colonies subjected to co‐cultivation after two weeks under antibiotic selection. Properly separated resistant growing points were chosen for plasmid rescue and subcultured for mycelia harvest. In order to evaluate the T‐DNA integration pattern of pHg/pBks 12 hygromycin‐resistant fungal strains were analysed for the presence of hph and amp transgenes by PCR. Only one of the 12 samples failed to show a signal for amp transgene (data not shown). In order to evaluate further the number and the pattern of integration events genomic DNA from 11 amp‐positive transgenic fungal strains and wild type was digested with BamHI, which cuts once within the T‐DNA (Fig. 1), Southern blotted and hybridized with amp probe (Fig. 2). Southern blot analysis suggested an integration pattern at variable genomic sites and dominantly as a single copy (10 out of 11 analysed transgenic strains with a single hybridization signal) confirming previous results with a different construct (Kemppainen et al., 2005). From this sample set we concluded that the 5.1 kb T‐DNA of the rescue‐plasmid pHg/pBks integrated mostly complete and as a single copy at variable sites in Laccaria genome. Genomic DNA of 51 fungal transgenic strains picked up at random from a larger collection (about 500) was extracted and checked by PCR for the presence of both transgenes (data not shown). Forty‐seven out of 51 transgenic strains were PCR‐positive for both amp and hph and were further analysed by RB plasmid rescue.


T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor.

Kemppainen M, Duplessis S, Martin F, Pardo AG - Microb Biotechnol (2008)

Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λBstE II, L. bicolor wild type (Wt) and transgenic strains.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815887&req=5

f2: Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λBstE II, L. bicolor wild type (Wt) and transgenic strains.
Mentions: Laccaria bicolor dikaryotic strain S238N (heterokaryotic, the phase in the fungal life cycle which forms symbiosis with tree roots) was transformed with Agrobacterium tumefaciens AGL‐1 carrying the pHg/pBks vector. The transformation efficiency with the given plasmid was 75% calculated from the appeared hygromycin resistant growing points against the total number of fungal colonies subjected to co‐cultivation after two weeks under antibiotic selection. Properly separated resistant growing points were chosen for plasmid rescue and subcultured for mycelia harvest. In order to evaluate the T‐DNA integration pattern of pHg/pBks 12 hygromycin‐resistant fungal strains were analysed for the presence of hph and amp transgenes by PCR. Only one of the 12 samples failed to show a signal for amp transgene (data not shown). In order to evaluate further the number and the pattern of integration events genomic DNA from 11 amp‐positive transgenic fungal strains and wild type was digested with BamHI, which cuts once within the T‐DNA (Fig. 1), Southern blotted and hybridized with amp probe (Fig. 2). Southern blot analysis suggested an integration pattern at variable genomic sites and dominantly as a single copy (10 out of 11 analysed transgenic strains with a single hybridization signal) confirming previous results with a different construct (Kemppainen et al., 2005). From this sample set we concluded that the 5.1 kb T‐DNA of the rescue‐plasmid pHg/pBks integrated mostly complete and as a single copy at variable sites in Laccaria genome. Genomic DNA of 51 fungal transgenic strains picked up at random from a larger collection (about 500) was extracted and checked by PCR for the presence of both transgenes (data not shown). Forty‐seven out of 51 transgenic strains were PCR‐positive for both amp and hph and were further analysed by RB plasmid rescue.

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.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Micología Molecular, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 352, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina.

Show MeSH
Related in: MedlinePlus