Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the amino-terminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.

译文

荧光蛋白标记被广泛用于报告植物膜运输; 然而,缺乏定量荧光或标记表达的有效方案。在这里,来自口蹄疫病毒的20个残基自切割2A肽被用于构建多蛋白,该多蛋白以固定的化学计量与参考蛋白在不同的细胞区室中表达了被贩运的标记。同时针对各种隔室。与定制的图像分析工具一起,这些构建体可以使用与普通植物转染和转基因系统兼容的协议,以显着提高的灵敏度,动态范围和统计意义来分析生物合成膜的流量。由于可以在群体或单个细胞中监测标记物和效应子的表达,因此可以避免饱和现象,并且可以控制随机或表观遗传影响。令人惊讶的是,对比率测定构建体的突变分析表明,2A肽对于有效切割携带单个内部信号肽的多蛋白是必不可少的,而信号肽是必不可少的。相反,带有两个信号肽/锚的构建体需要2A才能有效分离和稳定性,但是2A导致这种融合物的氨基末端部分被错误分选到液泡中。提出了一个模型来说明植物在这些研究和其他研究中的2A行为。

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