Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora
Abstract A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a ho...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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SpringerOpen
2019-06-01
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| Series: | AMB Express |
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| Online Access: | http://link.springer.com/article/10.1186/s13568-019-0818-1 |
| _version_ | 1818303967040372736 |
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| author | Yoichi Honda Eiji Tanigawa Takahisa Tsukihara Dong Xuan Nguyen Harunori Kawabe Naofumi Sakatoku Junko Watari Hideaki Sato Shigekazu Yano Takashi Tachiki Toshikazu Irie Takahito Watanabe Takashi Watanabe |
| author_facet | Yoichi Honda Eiji Tanigawa Takahisa Tsukihara Dong Xuan Nguyen Harunori Kawabe Naofumi Sakatoku Junko Watari Hideaki Sato Shigekazu Yano Takashi Tachiki Toshikazu Irie Takahito Watanabe Takashi Watanabe |
| author_sort | Yoichi Honda |
| collection | DOAJ |
| description | Abstract A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants. |
| first_indexed | 2024-12-13T06:03:12Z |
| format | Article |
| id | doaj.art-8937464974e746fdb840f3c5d51a9def |
| institution | Directory Open Access Journal |
| issn | 2191-0855 |
| language | English |
| last_indexed | 2024-12-13T06:03:12Z |
| publishDate | 2019-06-01 |
| publisher | SpringerOpen |
| record_format | Article |
| series | AMB Express |
| spelling | doaj.art-8937464974e746fdb840f3c5d51a9def2022-12-21T23:57:16ZengSpringerOpenAMB Express2191-08552019-06-019111210.1186/s13568-019-0818-1Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermisporaYoichi Honda0Eiji Tanigawa1Takahisa Tsukihara2Dong Xuan Nguyen3Harunori Kawabe4Naofumi Sakatoku5Junko Watari6Hideaki Sato7Shigekazu Yano8Takashi Tachiki9Toshikazu Irie10Takahito Watanabe11Takashi Watanabe12Graduate School of Agriculture, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityGraduate School of Agriculture, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityGraduate School of Life Sciences, Ritsumeikan UniversityBiotechnology Center of Ho Chi Minh CityEnvironmental Science Graduate School, The University of Shiga PrefectureResearch Institute for Sustainable Humanosphere, Kyoto UniversityResearch Institute for Sustainable Humanosphere, Kyoto UniversityAbstract A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.http://link.springer.com/article/10.1186/s13568-019-0818-1BasidiomyceteGenetic transformationTransient gene expressionPromoter assayTATAA sequence |
| spellingShingle | Yoichi Honda Eiji Tanigawa Takahisa Tsukihara Dong Xuan Nguyen Harunori Kawabe Naofumi Sakatoku Junko Watari Hideaki Sato Shigekazu Yano Takashi Tachiki Toshikazu Irie Takahito Watanabe Takashi Watanabe Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora AMB Express Basidiomycete Genetic transformation Transient gene expression Promoter assay TATAA sequence |
| title | Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora |
| title_full | Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora |
| title_fullStr | Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora |
| title_full_unstemmed | Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora |
| title_short | Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora |
| title_sort | stable and transient transformation and a promoter assay in the selective lignin degrading fungus ceriporiopsis subvermispora |
| topic | Basidiomycete Genetic transformation Transient gene expression Promoter assay TATAA sequence |
| url | http://link.springer.com/article/10.1186/s13568-019-0818-1 |
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