A 14-bp Stretch Plays a Critical Role in Regulating Gene Expression from β-tubulin Promoters of Basidiomycetes

Cis-acting elements play a vital role in regulation of transcription initiation. Several cis-acting elements have been identified in filamentous fungi; however, the fundamental requirements for basic promoter function in basidiomycetes are obscure. In this study, core elements in β-tubulin promoters of basidiomycetes were functionally characterized. Using transient transformation in Ceriporiopsis subvermispora as a promoter assay, we found that a 14-bp region (β-tubulin core promoter element, BCE), as well as CT-rich stretch, in the β-tubulin promoter of the species played a critical role in the expression of a recombinant hph as a reporter gene. In addition, in silico analysis revealed other members of basidiomycetes also harboured the BCE motif as well as CT-rich stretch in the β-tubulin promoter region, suggesting their functional conservation among the species of basidiomycetes. To confirm the function of BCE, we investigated the effects of BCE motif deletion in the Pleurotus ostreatus β-tubulin promoter on expression levels of a recombinant luminous shrimp luciferase reporter gene, which was targeted into the Pofcy1 locus. Intriguingly, luciferase activity was abolished when the BCE motif was deleted in the β-tubulin promoter, strongly demonstrating its essential function in transcription from this promoter on the chromosome. This study clearly demonstrates the crucial role of the BCE as well as the CT-rich stretch regions in the β-tubulin promoter among basidiomycetes and provides new insights into the fundamental mechanism of transcription initiation in this group.