Laboratory of Virology, Centre for Plant Virus Research, Institute of Experimental Botany of the Czech Academy of Sciences, 16500 Prague, Czech Republic.
Department of Plant Protection, Czech University of Life Sciences, 16500 Prague, Czech Republic.
Viruses. 2022 May 20;14(5):1099. doi: 10.3390/v14051099.
We have developed a Potato virus X (PVX)-based vector system compatible with the GoldenBraid 2.0 (GB) cloning strategy to transiently express heterologous proteins or peptides in plants for biotechnological purposes. This vector system consists of three domestication vectors carrying three GB parts-the cauliflower mosaic virus (CaMV) 35S promoter with PVX upstream of the second subgenomic promoter of the PVX coat protein (PVX CP SGP), nopaline synthase (NOS) terminator with PVX downstream of the first PVX CP SGP and the gene of interest (GOI). The full-length PVX clone carrying the sequence encoding a green fluorescent protein (GFP) as GOI was incorporated into the binary GB vector in a one-step reaction of three GB parts using the four-nucleotide GB standard syntax. We investigated whether the obtained vector named GFP/pGBX enables systemic PVX infection and expression of GFP in plants. We show that this GB-compatible vector system can be used for simple and efficient assembly of PVX-based expression constructs and that it meets the current need for interchange of standard biological parts used in different expression systems.
我们开发了一种基于马铃薯 X 病毒 (PVX) 的载体系统，与 GoldenBraid 2.0 (GB) 克隆策略兼容，可用于在植物中瞬时表达异源蛋白或肽，以实现生物技术目的。该载体系统由三个驯化载体组成，携带三个 GB 元件——PVX 外壳蛋白 (PVX CP) 第二个亚基因组启动子上游的花椰菜花叶病毒 (CaMV) 35S 启动子、PVX 第一个 CP SGP 下游的胭脂碱合成酶 (NOS) 终止子和感兴趣的基因 (GOI)。全长携带编码绿色荧光蛋白 (GFP) 的序列的 PVX 克隆被用作 GOI 被整合到二元 GB 载体中，在三步反应中使用四个核苷酸的 GB 标准语法整合三个 GB 元件。我们研究了命名为 GFP/pGBX 的获得载体是否能够在植物中引起系统的 PVX 感染和 GFP 的表达。我们表明，这种与 GB 兼容的载体系统可用于简单高效地组装基于 PVX 的表达构建体，并且满足当前在不同表达系统中使用标准生物部件互换的需求。
