ostreatus. To develop a system for in vivo analysis of poxa1b promoter and its metal regulation, the gene-encoding GFP was adopted as reporter gene putting its expression under the control of 1400-bp-long poxa1b promoter region. GFP of the jellyfish Aequorea victorea emits fluorescence as a result of its intrinsic chromophore structure, not requiring any substrate or cofactor (Chalfie et al., 1994), and it represents a versatile reporter gene (Cubitt et al., 1995). The vector pEGFPea1b for in vivo analysis of P. ostreatus laccase promoters was constructed using the gene coding for

enhanced GFP (EGFP). A P. ostreatus poxa1b promoter region of 1336 bp was used as cis-regulatory element to drive expression of EGFP. An intron/exon fragment containing an intron/exon sequence of the poxc MG-132 clinical trial gene was included between the poxa1b promoter and the egfp gene, considering previous results showing that efficient GFP expression in Agaricus bisporus and Coprinus cinereus (Burns et al., 2005) and Phanerochaete chrysosporium (Ma et al., 2001) requires introns. A homologous selection marker, the mutant gene cassette CbxR, encoding a modified iron–sulfur protein Ip subunit of succinate dehydrogenase with an aminoacid substitution (His239 to Leu) and conferring

resistance to systemic fungicide carboxin (Honda et al., 2000), was adopted. Cotransformation with pTM1 vector conferring carboxin resistance and pEGFPea1b vector containing egfp gene under the control of poxa1b promoter region was carried out, by adopting check details an adapted version of transformation protocol reported by Salame et al. (2010). Moreover, an unique vector containing both the mutant gene cassette CbxR and the reporter cassette poxa1b promoter-egfp Celecoxib gene was constructed and adopted for transformation. Transformants were firstly screened for carboxin resistance. The carboxin-resistant colonies were subjected to at least four rounds

of selection by transferring on fresh selection medium. Around 50 carboxin-resistant transformants were obtained per μg of pTM1 DNA per 107 viable protoplasts in a transformation with pTM1 and pEGFPea1b, and five carboxin-resistant transformants were obtained per μg of pEGFPCBX DNA per 107 viable protoplasts in a transformation with this vector. Hence, cotransformation with vectors containing gene cassette CbxR and the reporter cassette poxa1b promoter-egfp gene allowed a 10-fold higher transformation efficiency than transformation with an unique vector containing both cassettes. This could be ascribed to the larger size of the latter construct. The carboxin-resistant transformants were further analyzed for checking the presence of egfp and fluorescence emission. Carboxin-resistant transformants were analyzed by PCR to verify the presence of the transforming DNA.