High efficient homologous integration via tandem exo-beta-1,3-glucanase genes in the common mushroom, Agaricus bisporus

• Published in: Current genetics 1996, Vol.30 (2), p.166-173
• Main Authors: Rhee, M.D. van de, Mendes, O, Werten, M.W.T, Huizing, H.J, Mooibroek, H
• Format: Article
• Language: English
• Subjects: abexg1 gene; abexg2 gene; Agaricus bisporus; amino acid sequences; codon usage; fruiting bodies; fungal anatomy; genbank/x92961; gene expression; genetic code; genetic transformation; homologous recombination; introns; linearized plasmids; messenger RNA; molecular sequence data; mycelium; nucleotide sequences; O-glycoside hydrolases; open reading frames; plasmid vectors; restriction mapping; structural genes
• ISSN: 0172-8083; 1432-0983

Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30-60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5'- or 3'-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-beta-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology.