Investigating dominant selection markers for Coprinopsis cinerea: a carboxin resistance system and re-evaluation of hygromycin and phleomycin resistance vectors
Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinereasdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful. Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant (ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance, where introns are not required for expression, emphasising the variability in importance of these elements.
KeywordsFungicide resistance Basidiomycete Transformation Intron Homologous marker gene Dominant selectable marker genes
- Challen MP, Gregory KE, Sreenivasaprasad S, Rogers CC, Cutler SB, Diaper DC, Elliot TJ, Foster GD (2000) Transformation technologies for mushrooms. Mushroom Sci 15:165–172Google Scholar
- Kües U, Richardson WV, Tymon AM, Mutasa ES, Göttgens B, Gaubatz S, Gregoriades A, Casselton LA (1992) The combination of dissimilar alleles of the Aα and Aβ gene complexes, whose proteins contain homeo domain motifs, determines sexual development in the mushroom Coprinus cinereus. Genes Dev 6:568–577CrossRefPubMedGoogle Scholar
- Ogawa K, Yamazaki T, Hasebe T, Kajiwara S, Watanabe A, Asada Y, Shishido K (1998) Molecular breeding of the basidiomycete Coprinus cinereus strains with high lignin-decolorization and -degradation activities using novel heterologous protein expression vectors. Appl Microbiol Biotechnol 49:285–289CrossRefPubMedGoogle Scholar
- Sambrook J, Russell DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar