Functional analysis of gene expression signals in basidiomycetous fungi using transfection, and random or targeted integration on the chromosome
Gene expression signals such as promoter and terminator sequences play an essential role in controlling timing and efficiency of each gene’s expression. In basidiomycetes, basic requirements for gene expression signals are obscure because of the lack of proper experimental approaches. In this report, a functional analysis of promoter sequence was conducted in white-rot basidiomycete, Ceriporiopsis subvermispora, using transfection phenomena with recombinant hygromycin phosphotransferase (hph) genes as a reporter. We found that a 14-bp region (BCE) plays a critical role in expression of the recombinant hph driven by the promoter sequence of ß-tubulin gene in C. subvermispora. In silico analysis demonstrated that other basidiomycetes also harboured BCE-like sequence in ß-tubulin promoter, suggesting its functional conservation among species of the class. To address this hypothesis, we investigated the effects of deletion of the BCE-like sequence in ß-tubulin promoter on expression levels of the reporter genes, hph or NanoLuci, in Pleurotus ostreatus and Coprinopsis cinerea. The reporter genes driven by either deletion mutant of BCE-like sequence or intact ß-tubulin promoter from each species was integrated into host chromosome either randomly or targeted site by homologous recombination. The results demonstrated its essential function in transcription from these promoters. Our findings provide new insights into the vital role of BCE region in the ß-tubulin promoter among basidiomycetes. The transfection system was also used for characterizing sequence involved in 3’ end formation of mRNA of glyceraldehyde-3-phosphate dehydrogenase (gpd) terminator in C. subvermispora. Our results showed that an AT-rich region located at 13 nt upstream of the polyA site played a crucial role in the function of gpd terminator. It is conceivable that the assay system developed in this work is available for analysis of other functional sequences like translation controlling signals etc.