DNA sequences of 2A viral peptides.

The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.社会变形虫盘基网柄菌（Dictyostelium discoideum）是多种生物学过程的模式生物，涵盖趋化性、细胞间通讯、吞噬作用及发育进程。利用现代遗传学工具研究这些过程，通常需要表达多种转基因（transgene）。尽管可同时转染多个转录单元（transcriptional unit），但为每个基因使用独立的启动子（promoter）和终止子（terminator）会导致质粒（plasmid）体积过大，且单元间可能发生相互干扰。在诸多真核系统（eukaryotic system）中，这一难题可通过由2A病毒肽（2A viral peptides）介导的多顺反子表达（polycistronic expression）策略解决，从而实现高效且共调控的基因表达。本研究针对盘基网柄菌，筛选了当前最常用的四种2A病毒肽：猪捷申病毒1型2A（P2A）、泰萨病毒2A（T2A）、马鼻炎A病毒2A（E2A）以及口蹄疫病毒2A（F2A）的活性，结果显示所有受试2A序列均具有活性。然而，将两种蛋白质的编码序列整合至单个转录本（transcript）中，会引发显著的菌株依赖性表达水平下降，这表明盘基网柄菌体内存在其他调控基因表达的因子，有待进一步探究。本研究结果证实，P2A是盘基网柄菌中多顺反子表达的最优序列，为该模式生物的基因工程研究开辟了全新可能。