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Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens.

Sharma KK, Kuhad RC - BMC Biotechnol. (2010)

Bottom Line: The fungal transformants were confirmed by PCR and Southern hybridization.The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.

ABSTRACT

Background: White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.

Results: This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.

Conclusion: These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

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Related in: MedlinePlus

Molecular screening of transformants. (a). PCR of transformed mycelia using GUS-GFP fusion primer. Lane M: DNA molecular weight marker (kb); Lane 1&2: Ganoderma sp. RCKK-02; Lane 3&4: P. cinnabarinus; Lane 5&6: Crinipellis sp. RCK-1; Lane 7&8: P. sojur-caju; Lane 9&10: P. chrysosporium; Lane 11&12: Fungal isolate BHR-UDSC. Lanes 2,4,6,8,10 & 12 are acetosyringone (AS) pre-induced, whereas others are without AS. The Lane C1 & Lane C2 were positive control and negative control, respectively. (b). Southern blot analysis of transformed fungus using radiolabelled hpt gene probe. Lane WT: Untransformed; Lane A: Ganoderma sp. RCKK-02; Lane B: P. cinnabarinus; Lane C: Crinipellis sp. RCK-1; Lane D: P. sojur-caju; Lane E: Positive control; Lane F: P. chrysosporium; Lane G: Fungal isolate BHR-UDSC. (c). Southern blot analysis of different transformants using KanR probe. Lane C: Untransformed control; Lane 1: Ganoderma sp. RCKK-02; Lane 2: P. cinnabarinus; Lane 3: Crinipellis sp. RCK-1; Lane 4: P. sojur-caju; Lane 5: P. chrysosporium; Lane 6: Fungal isolate BHR-UDSC.
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Figure 5: Molecular screening of transformants. (a). PCR of transformed mycelia using GUS-GFP fusion primer. Lane M: DNA molecular weight marker (kb); Lane 1&2: Ganoderma sp. RCKK-02; Lane 3&4: P. cinnabarinus; Lane 5&6: Crinipellis sp. RCK-1; Lane 7&8: P. sojur-caju; Lane 9&10: P. chrysosporium; Lane 11&12: Fungal isolate BHR-UDSC. Lanes 2,4,6,8,10 & 12 are acetosyringone (AS) pre-induced, whereas others are without AS. The Lane C1 & Lane C2 were positive control and negative control, respectively. (b). Southern blot analysis of transformed fungus using radiolabelled hpt gene probe. Lane WT: Untransformed; Lane A: Ganoderma sp. RCKK-02; Lane B: P. cinnabarinus; Lane C: Crinipellis sp. RCK-1; Lane D: P. sojur-caju; Lane E: Positive control; Lane F: P. chrysosporium; Lane G: Fungal isolate BHR-UDSC. (c). Southern blot analysis of different transformants using KanR probe. Lane C: Untransformed control; Lane 1: Ganoderma sp. RCKK-02; Lane 2: P. cinnabarinus; Lane 3: Crinipellis sp. RCK-1; Lane 4: P. sojur-caju; Lane 5: P. chrysosporium; Lane 6: Fungal isolate BHR-UDSC.

Mentions: The Hygr transformants were PCR screened with gus-gfp primers which resulted in an expected amplified product of 2.5 kb (Figure 5a). Randomly selected transformants were further used for molecular analysis. The BamHI digested genomic DNA from transformants was hybridized to P32 α dATP labelled htp. The Southern analysis confirmed transformation and also revealed that the number of inserts in different transformed lines varied from one to four (Figure 5b). All the Hygr mycelia were tested for the possibility of Agrobacterium contamination. Fungal mycelia were grown on LB medium to screen any bacterial contamination. The BamHI digested genomic DNA samples from the transformed lines were analysed by Southern hybridization using KanR probe. No hybridization was detected in the transformants, which ruled out the possibility of any bacterial contamination (Figure 5c).


Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens.

Sharma KK, Kuhad RC - BMC Biotechnol. (2010)

Molecular screening of transformants. (a). PCR of transformed mycelia using GUS-GFP fusion primer. Lane M: DNA molecular weight marker (kb); Lane 1&2: Ganoderma sp. RCKK-02; Lane 3&4: P. cinnabarinus; Lane 5&6: Crinipellis sp. RCK-1; Lane 7&8: P. sojur-caju; Lane 9&10: P. chrysosporium; Lane 11&12: Fungal isolate BHR-UDSC. Lanes 2,4,6,8,10 & 12 are acetosyringone (AS) pre-induced, whereas others are without AS. The Lane C1 & Lane C2 were positive control and negative control, respectively. (b). Southern blot analysis of transformed fungus using radiolabelled hpt gene probe. Lane WT: Untransformed; Lane A: Ganoderma sp. RCKK-02; Lane B: P. cinnabarinus; Lane C: Crinipellis sp. RCK-1; Lane D: P. sojur-caju; Lane E: Positive control; Lane F: P. chrysosporium; Lane G: Fungal isolate BHR-UDSC. (c). Southern blot analysis of different transformants using KanR probe. Lane C: Untransformed control; Lane 1: Ganoderma sp. RCKK-02; Lane 2: P. cinnabarinus; Lane 3: Crinipellis sp. RCK-1; Lane 4: P. sojur-caju; Lane 5: P. chrysosporium; Lane 6: Fungal isolate BHR-UDSC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Molecular screening of transformants. (a). PCR of transformed mycelia using GUS-GFP fusion primer. Lane M: DNA molecular weight marker (kb); Lane 1&2: Ganoderma sp. RCKK-02; Lane 3&4: P. cinnabarinus; Lane 5&6: Crinipellis sp. RCK-1; Lane 7&8: P. sojur-caju; Lane 9&10: P. chrysosporium; Lane 11&12: Fungal isolate BHR-UDSC. Lanes 2,4,6,8,10 & 12 are acetosyringone (AS) pre-induced, whereas others are without AS. The Lane C1 & Lane C2 were positive control and negative control, respectively. (b). Southern blot analysis of transformed fungus using radiolabelled hpt gene probe. Lane WT: Untransformed; Lane A: Ganoderma sp. RCKK-02; Lane B: P. cinnabarinus; Lane C: Crinipellis sp. RCK-1; Lane D: P. sojur-caju; Lane E: Positive control; Lane F: P. chrysosporium; Lane G: Fungal isolate BHR-UDSC. (c). Southern blot analysis of different transformants using KanR probe. Lane C: Untransformed control; Lane 1: Ganoderma sp. RCKK-02; Lane 2: P. cinnabarinus; Lane 3: Crinipellis sp. RCK-1; Lane 4: P. sojur-caju; Lane 5: P. chrysosporium; Lane 6: Fungal isolate BHR-UDSC.
Mentions: The Hygr transformants were PCR screened with gus-gfp primers which resulted in an expected amplified product of 2.5 kb (Figure 5a). Randomly selected transformants were further used for molecular analysis. The BamHI digested genomic DNA from transformants was hybridized to P32 α dATP labelled htp. The Southern analysis confirmed transformation and also revealed that the number of inserts in different transformed lines varied from one to four (Figure 5b). All the Hygr mycelia were tested for the possibility of Agrobacterium contamination. Fungal mycelia were grown on LB medium to screen any bacterial contamination. The BamHI digested genomic DNA samples from the transformed lines were analysed by Southern hybridization using KanR probe. No hybridization was detected in the transformants, which ruled out the possibility of any bacterial contamination (Figure 5c).

Bottom Line: The fungal transformants were confirmed by PCR and Southern hybridization.The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, India.

ABSTRACT

Background: White-rot fungi are primarily the major degraders of lignin, a major obstacle for commercial exploitation of plant byproducts to produce bioethanol and other industrially important products. However, to improve their efficacy for lignin degradation, it has become necessary to genetically modify these organisms using appropriate vectors. Agrobacterium tumefaciens, a soil phytopathogenic bacterium, generally transforms plants by delivering a portion of the resident Ti- plasmid, the T-DNA (transfer DNA). The trans-Kingdom gene transfer is initiated by the activity of Ti-plasmid encoded vir (virulence) genes in response to low-molecular-mass phenolic compounds such as acetosyringone. A. tumefaciens played a major role in plant genetic engineering and basic research in molecular biology, accounting for nearly 80% of the transgenic plants produced so far. Initially, it was believed that only dicotyledons, gymnosperms and a few monocotyledonous species could be transformed by this bacterium; but recent reports have totally changed this scenario by demonstrating that many 'recalcitrant' species not included in its natural host range can also be transformed, especially filamentous fungi.

Results: This paper describes an efficient and convenient Agrobacterium-mediated gene transformation system for successful delivery of T-DNA, carrying the genes coding for β-glucuronidase (uidA), green fluorescent protein (gfp) and hygromycin phosphotransferase (hpt) to the nuclear genome of lignin degrading white-rot fungi such as Phanerochaete chrysosporium, Ganoderma sp. RCKK-02, Pycnoporous cinnabarinus, Crinipellis sp. RCK-1, Pleurotus sajor-caju and fungal isolate BHR-UDSC without supplementation of acetosyringone. The fungal transformants were confirmed by PCR and Southern hybridization. The expression vector pCAMBIA 1304-RCKK was constructed by the addition of GPD promoter from plasmid p416 to the binary vector backbone pCAMBIA1304, which controls uidA and gfp gene. Transmission Electron Microscope (TEM) analysis revealed the attachment of bacterial cells to the fungal hyphae. Transformation frequency varied from 50 to 75% depending on the fungal species used in this study. The transformation efficiency was maximum at 20°C whereas no transfer was observed at temperature above 29°C.

Conclusion: These findings provide a rapid and reproducible transformation method without external addition of acetosyringone, which could be useful for improving white-rot fungi for their various biotechnological applications.

Show MeSH
Related in: MedlinePlus