Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624 |
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Authors: | Yamagishi Kenji Kimura Toshiyuki Oita Sigeru Sugiura Tatsuki Hirai Hirofumi |
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Institution: | (1) National Agricultural Research Center for the Tohoku Region, National Agriculture and Food Research Organization, Arai Fukushima, 960-2156, Japan;(2) Faculty of Agriculture, Shizuoka University, Ohya 836, Suruga-ku Shizuoka, 422-8529, Japan |
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Abstract: | Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance
as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid
pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 × 107 protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene
was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions. |
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Keywords: | Phanerochaete sordida EGFP Genetic transformation PsURA5 Uracil auxotroph |
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