The use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labeling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

荧光蛋白的使用允许从活体成像和固定细胞到标记整个生物体的多种方法，使其成为各种实验的基础。标记感兴趣的蛋白质或特定细胞类型允许对细胞定位、细胞动力学、生理学和结构特征进行可视化和研究。在特定情况下，荧光融合蛋白可能由于阻碍蛋白质功能的结构变化而无法正常发挥功能，或者当过度表达时可能具有细胞毒性并破坏正常的生物过程。在我们的研究中，我们描述了双顺反子载体的应用，该载体在NAT抗生素选择标记和 mCherry。这允许从单个开放阅读框表达多个基因，并通过 2A 肽内的切割事件产生离散的蛋白质产物。我们展示了将这个双顺反子向量集成到模型中马拉色菌物种，单倍体菌株M.furfurCBS 14141，具有主动选择、高荧光和经过验证的蛋白水解切割。该技术的潜在应用包括蛋白质功能研究，马拉色菌细胞定位和靶向诱变所需基因的共表达。