Characterization of the effects of terminators and introns on recombinant gene expression in the basidiomycete Ceriporio

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eISSN 1976-3794 pISSN 1225-8873

Characterization of the effects of terminators and introns on recombinant gene expression in the basidiomycete Ceriporiopsis subvermispora§ Dong Xuan Nguyen1,2, Emi Nishisaka1, Moriyuki Kawauchi1, Takehito Nakazawa1, Masahiro Sakamoto1, and Yoichi Honda1* 1

Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan 2 Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Viet Nam (Received Apr 20, 2020 / Revised Aug 4, 2020/ Accepted Aug 5, 2020)

Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csβ1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class. Keywords: Ceriporiopsis subvermispora, transient transformation, gpd gene, polyadenylation signals, intron functions Introduction Determining the fundamental requirements for gene expression in filamentous fungi is not only indispensable for further characterizing the associated regulatory mechanism but also for enabling the efficient expression of homologous or heterologous genes in fungi. Regarding ascomycetous fungi, there has been considerable research aimed at identifying the fundamental factors controlling gene expression, including high-capacity terminators, strong promoters, the presence *For correspondence. Email: [email protected]; Tel.: +8175-753-6463; Fax: +81-75-753-6471 § Supplemental material for this article may be found at http://www.springerlink.com/content/120956. Copyright ⓒ 2020, The Microbiological Society of Korea

of introns, and the protein secretion capacity as well as the protease deficiency of expression host strains (Peterson and Nevalainen, 2012; Curran et al., 2013; Ito et al., 2013; Landowski et al., 2015; Hoshida et al., 2017; Nambu-Nishida et al., 2018). In contrast, in basidiomycetes, the essential factors for efficient gene expression remain poorly understood. A recent analysis of ascomycete and basidiomycete genomes revealed a significant difference in the regulatory repertoires between these two phyla (Todd et al., 2014). In this context, regulating gene expressio