Yoon, J.; Kim, Y.; Kim, S.; Jeong, H.; Park, J.; Jeong, M.-H.; Park, S.; Jo, M.; An, S.; Park, J.; Jang, S.-H.; Goh, J.; Park, S.-Y. Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546. J. Fungi2023, 9, 1158.
Yoon, J.; Kim, Y.; Kim, S.; Jeong, H.; Park, J.; Jeong, M.-H.; Park, S.; Jo, M.; An, S.; Park, J.; Jang, S.-H.; Goh, J.; Park, S.-Y. Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546. J. Fungi 2023, 9, 1158.
Yoon, J.; Kim, Y.; Kim, S.; Jeong, H.; Park, J.; Jeong, M.-H.; Park, S.; Jo, M.; An, S.; Park, J.; Jang, S.-H.; Goh, J.; Park, S.-Y. Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546. J. Fungi2023, 9, 1158.
Yoon, J.; Kim, Y.; Kim, S.; Jeong, H.; Park, J.; Jeong, M.-H.; Park, S.; Jo, M.; An, S.; Park, J.; Jang, S.-H.; Goh, J.; Park, S.-Y. Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546. J. Fungi 2023, 9, 1158.
Abstract
Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for functional gene analysis. We generated 3,689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation time of fungal spores and Agrobacterium tumefaciens cells was performed at 24–36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using ATMT, co-cultivation was reduced to ≤ 36 h. The resulting transformants were mitotically stable, and PCR analysis confirmed gene integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed by PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analysis to study genes of interest in P. inflatum.
Biology and Life Sciences, Biology and Biotechnology
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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