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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for <t>S.</t> <t>rugosoannulata</t> mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto <t>PDA</t> medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.
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Establishment of an ATMT system for S. rugosoannulata mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto PDA medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.

Journal: Journal of Fungi

Article Title: Unlocking the Potential for Genetic Engineering of the Straw-Degrading Mushroom Stropharia rugosoannulata by Constructing a CRISPR/Cas9 Gene Editing System

doi: 10.3390/jof12040269

Figure Lengend Snippet: Establishment of an ATMT system for S. rugosoannulata mycelium. ( A ) The GPiE-SrU6 plasmid carrying two knockout targets was introduced into A. tumefaciens GV3101 for amplification, followed by cocultivation of wounded mycelium with Agrobacterium for 96 h. ( B ) After cocultivation, the mycelium was washed in medium containing 600 mg/L Cef, inoculated onto PDA medium supplemented with 60 µg/mL Hyg for primary screening, and subsequently transferred onto PDA medium containing 0.2 mg/mL 5-FOA for secondary screening. Growth on different selective drugs (Cef, Hyg and 5-FOA), with three biological replicates for each treatment. ( C ) Gel electrophoresis image of the hygromycin gene amplified from DNA extracted from some candidate transgenic strains.

Article Snippet: Furthermore, S. rugosoannulata was inoculated onto PDA resistance medium supplemented with 0, 0.05, 0.1, 0.2, or 0.3 mg/mL 5-FOA (Solarbio Science & Technology Co., Ltd., Beijing, China).

Techniques: Plasmid Preparation, Knock-Out, Amplification, Nucleic Acid Electrophoresis, Transgenic Assay

Growth phenotypes of mutant strains on different culture media and editing efficiency of the ura3 gene. ( A ) Growth phenotypes of WT and mutant strains on MM, PDAU (PDA medium supplemented with 5 mM uracil), and PDAU+5-FOA (PDAU supplemented with 0.2 mg/mL 5-FOA). The WT and mutant strains were grown on different culture media, with three biological replicates per treatment. ( B ) Analysis of the editing efficiency of the ura3 gene in S. rugosoannulata using the CRISPR/Cas9 system.

Journal: Journal of Fungi

Article Title: Unlocking the Potential for Genetic Engineering of the Straw-Degrading Mushroom Stropharia rugosoannulata by Constructing a CRISPR/Cas9 Gene Editing System

doi: 10.3390/jof12040269

Figure Lengend Snippet: Growth phenotypes of mutant strains on different culture media and editing efficiency of the ura3 gene. ( A ) Growth phenotypes of WT and mutant strains on MM, PDAU (PDA medium supplemented with 5 mM uracil), and PDAU+5-FOA (PDAU supplemented with 0.2 mg/mL 5-FOA). The WT and mutant strains were grown on different culture media, with three biological replicates per treatment. ( B ) Analysis of the editing efficiency of the ura3 gene in S. rugosoannulata using the CRISPR/Cas9 system.

Article Snippet: Furthermore, S. rugosoannulata was inoculated onto PDA resistance medium supplemented with 0, 0.05, 0.1, 0.2, or 0.3 mg/mL 5-FOA (Solarbio Science & Technology Co., Ltd., Beijing, China).

Techniques: Mutagenesis, CRISPR