Development of High Efficiency Transformation System Using Retrotransposon in Basidiomyceteous Fungi

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To understand an unique biological phenomenon of basidiomycete fungi and produce basidiomycete strains useful for recycle of plant biomass resource and preservation of environment., it is necessary to develop the system of high efficiency gene introduction and expression. By using this system, we can easily select the desired strain. We used LTR (long terminal repeat) of retrotransposon Le.RTn1 isolated from Lentinula edodes chromosome as a promoter element from the following reason : Le.RTn1 (6,213 bp) contained two internal overlapping open reading frames (ORFs) putatively encoding GAG (group-specific antigens), and PRO (protease) plus POL (reverse transcriptase, RNase H, and integrase). The LTR (474 bp) contained four consecutive eukaryotic promoter consensus TATA(like)-boxes, and three relatively long direct repeats that are present upstream, within or downstream of the TATA(like)-boxes. We cloned the L.edodes cDNA (641 bp) encoding a cytochrome P450 heme-binding domain (SHP1 : small heme-binding protein 1), to 5' end of which the part of the LTR sequence is fused. The 5' end (corresponding to the transcription start point) of the cloned cDNA was mapped to the position just downstream of the most downstream TATA box. L.edodes mycelial cells contained a large amount of shp1 transcript. These evidences show a strong promoter activity of the LTR. We constructed the chromosome-integrating recombinant plasmid containing the expression cassette of the LTR promoter-hygromycin-resistant gene-L.edodes priA terminator and introduced it into basidiomycete Coriolus hirsutus. The hygromycin-resistant colonies were obtained with a high frequency.