DNA-mediated transformation system in a bipolar basidiomycete, Pholiota microspora (P. nameko)
2009
Yi, R.(Tottori Univ. (Japan)) | Tachikawa, T. | Mukaiyama, H. | Mochida, Y. | Ishikawa, M. | Aimi, T.
We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/microg pMBsip2 DNA using 5 x 10E6 protoplasts in regeneration plates containing 1.0 microg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/microg pMBhph1 DNA using 5 x 10E6 protoplasts in regeneration plates containing 150 microg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.
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