Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Genetic Introduction of Foreign Genes to Pleurotus eryngii by Restriction Enzyme-Mediated Integration: Won Noh , Sang-Woo Kim , Dong-Won Bae , Jae-Yean Kim , Hyeon-Su Ro; J. Microbiol. 2010;48(2):253-256. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-9278-7

Abstract: Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 106 mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10-40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.

Transformation and Mutagenesis of the Nematode-trapping Fungus Monacrosporium sphaeroides by Restriction Enzyme-mediated Integration (REMI): Xu Jin , Ming-He Mo , Zhou Wei , Xiao-Wei Huang , Ke-Qin Zhang; J. Microbiol. 2005;43(5):417-423.; DOI: https://doi.org/2281 [pii]

Abstract: In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants g-1 per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

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