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Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

Mycobiology 2021년 49권 5호 p.491 ~ 497
정민혜, Kim Jung-A., 강석찬, Choi Eu-Ddeum, 김영민, Lee Ye-Rim, 전미진, Yu Nan-Hee, 박애란, 김진철, 김순옥, 박숙영,
소속 상세정보
정민혜 ( Jeong Min-Hye ) - Sunchon National University Department of Plant medicine
 ( Kim Jung-A. ) - National Institute of Biological Resources Microorganism Resources Division
강석찬 ( Kang Seog-Chan ) - Pennsylvania State University Department of Plant Pathology and Environmental Microbiology
 ( Choi Eu-Ddeum ) - Sunchon National University Department of Plant medicine
김영민 ( Kim Young-Min ) - Sunchon National University Department of Plant medicine
 ( Lee Ye-Rim ) - Sunchon National University Department of Plant medicine
전미진 ( Jeon Mi-Jin ) - National Institute of Biological Resources Microorganism Resources Division
 ( Yu Nan-Hee ) - Chonnam National University Department of Agricultural Chemistry
박애란 ( Park Ae-Ran ) - Chonnam National University Department of Agricultural Chemistry
김진철 ( Kim Jin-Cheol ) - Chonnam National University Department of Agricultural Chemistry
김순옥 ( Kim Soon-Ok ) - National Institute of Biological Resources Microorganism Resources Division
박숙영 ( Park Sook-Young ) - Sunchon National University Department of Plant medicine

Abstract


An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

키워드

ATMT; fungal transformation; gene manipulation; Xylaria grammica

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