Table_1_Expression of a Malassezia Codon Optimized mCherry Fluorescent Protein in a Bicistronic Vector.DOCX

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（fluorescent proteins）的应用可涵盖活细胞成像、固定细胞标记至完整生物体标记等多种研究手段，是各类实验的重要基础。对目标蛋白或特定细胞类型进行标记，可实现细胞定位、细胞动态变化、生理特性及结构特征的可视化观测与研究。但在特定场景中，荧光融合蛋白可能因结构改变而无法正常发挥功能；若过表达，则可能产生细胞毒性并破坏正常生物学过程。本研究中，我们介绍了一种双顺反子载体（bicistronic vector）的应用：该载体在NAT抗生素筛选标记（NAT antibiotic selection marker）与mCherry之间插入了小RNA病毒（Picornavirus）2A肽段序列。该载体可实现单个开放阅读框（open reading frame）同时表达多个基因，并通过2A肽段内的剪切事件生成独立的蛋白产物。我们证实，该双顺反子载体可成功整合至模式马拉色菌（Malassezia）的单倍体菌株糠秕马拉色菌（M. furfur）CBS 14141中，且筛选活性有效、荧光信号强劲，并已验证其蛋白剪切功能正常。该技术的潜在应用包括蛋白功能研究、马拉色菌细胞定位，以及靶向诱变所需基因的共表达。