Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants.
Once for All: A Novel Robust System for Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in Plants.
复制标题
一劳永逸：一种用于在植物中共表达多种嵌合荧光融合蛋白的新型稳健系统
DOI：
10.3389/fpls.2017.01071
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发表时间：
2017
影响因子：
5.6
通讯作者：
Wang H
中科院分区：
文献类型：
--
作者：
Zhong G;Zhu Q;Li Y;Liu Y;Wang H
嵌合荧光融合蛋白是研究活细胞中蛋白质亚细胞定位和动态变化的有力工具。荧光融合蛋白与已知的细胞器标记物在同一细胞中的共表达在揭示所讨论的蛋白质的空间和时间功能方面特别有用。然而，在植物中共表达多种荧光标记蛋白的常规方法具有表达效率低、表达水平变化和耗时的遗传杂交的缺点。在这里，我们已经开发了一种新的强大的系统，允许在植物中以节省时间的方式高效共表达多种嵌合荧光融合蛋白。该系统利用单一表达载体的优点，该表达载体由多个半独立表达盒组成，用于蛋白质共表达，从而克服了使用多个独立表达质粒的局限性。此外，它是一个高度可操作的DNA组装系统，其中DNA分子的修饰和重组可以通过优化的一步组装反应轻松实现。利用这一有效的系统，我们证明了两个嵌合的荧光融合报告蛋白的液泡分选受体和分泌载体膜蛋白的共表达引起了他们的前景在植物中的亚细胞定位通过瞬时表达和稳定转化。因此，我们认为，这一技术进步代表了一种有前途的方法，多色蛋白质共表达在植物细胞中。
Chimeric fluorescent fusion proteins have been employed as a powerful tool to reveal the subcellular localizations and dynamics of proteins in living cells. Co-expression of a fluorescent fusion protein with well-known organelle markers in the same cell is especially useful in revealing its spatial and temporal functions of the protein in question. However, the conventional methods for co-expressing multiple fluorescent tagged proteins in plants have the drawbacks of low expression efficiency, variations in the expression level and time-consuming genetic crossing. Here, we have developed a novel robust system that allows for high-efficient co-expression of multiple chimeric fluorescent fusion proteins in plants in a time-saving fashion. This system takes advantage of employing a single expression vector which consists of multiple semi-independent expressing cassettes for the protein co-expression thereby overcoming the limitations of using multiple independent expressing plasmids. In addition, it is a highly manipulable DNA assembly system, in which modification and recombination of DNA molecules are easily achieved through an optimized one-step assembly reaction. By employing this effective system, we demonstrated that co-expression of two chimeric fluorescent fusion reporter proteins of vacuolar sorting receptor and secretory carrier membrane protein gave rise to their perspective subcellular localizations in plants via both transient expression and stable transformation. Thus, we believed that this technical advance represents a promising approach for multi-color-protein co-expression in plant cells.