Aboulela Mostafa, Tanaka Yuji, Nishimura Kohji, Mano Shoji, Nishimura Mikio, Ishiguro Sumie, Kimura Tetsuya, Nakagawa Tsuyoshi
Department of Molecular and Functional Genomics, Interdisciplinary Center for Science Research, Organization for Research, Shimane University, Matsue, Japan.
Bioresources Science, The United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan.
PLoS One. 2017 May 16;12(5):e0177889. doi: 10.1371/journal.pone.0177889. eCollection 2017.
Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones. In this system, C-terminal fusions with 17 kinds of tags including visible reporters and epitope tags are available for each ORF, and selection by four kinds of resistance markers is possible. We verified that the R4DS Gateway cloning system functioned well in Arabidopsis thaliana by observing the expression and localization patterns of fluorescent proteins fused with organelle-targeting signals and driven by stomatal-lineage specific promoters. We also confirmed that the two cloning sites in the R4DS Gateway cloning system were equivalent and independently regulated. The results obtained indicate that the R4DS Gateway cloning system facilitates detailed comparisons of the expression patterns of two promoters as well as co-localization and interaction analyses of two proteins in specific cells in plants.
大量蛋白质协同发挥作用，在各种细胞中行使其功能。为了了解这些蛋白质在植物中的功能和分子机制，通常需要对同时表达两个蛋白质编码基因的转基因植物进行分析。我们开发了一种新型的与Gateway克隆技术兼容的二元载体系统，即R4双位点（R4DS）Gateway克隆系统，它能够使用启动子和开放阅读框（ORF）入门克隆，轻松高效地将两组所需的启动子和开放阅读框克隆到二元载体中。在该系统中，每个ORF都可以与包括可见报告基因和表位标签在内的17种标签进行C端融合，并且可以通过四种抗性标记进行筛选。我们通过观察与细胞器靶向信号融合并由气孔谱系特异性启动子驱动的荧光蛋白的表达和定位模式，验证了R4DS Gateway克隆系统在拟南芥中功能良好。我们还证实，R4DS Gateway克隆系统中的两个克隆位点是等效且独立调控的。所得结果表明，R4DS Gateway克隆系统有助于详细比较两个启动子的表达模式，以及植物特定细胞中两种蛋白质的共定位和相互作用分析。
