Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research.
Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research.
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DOI：
10.1371/journal.pone.0177889
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Nakagawa T
中科院分区：
文献类型：
--
作者：
Aboulela M;Tanaka Y;Nishimura K;Mano S;Nishimura M;Ishiguro S;Kimura T;Nakagawa T
大量的蛋白质协同工作，在各种细胞中发挥其功能。为了了解这些蛋白在植物中的功能和分子机制，通常需要分析同时表达两个蛋白编码基因的转基因植物。我们开发了一种新的网关克隆技术兼容的二元载体系统，R4双位点（R4 DS）网关克隆系统，它能够使用启动子和开放阅读框（ORF）进入克隆将两组所需的启动子和ORF轻松有效地克隆到二元载体中。在该系统中，每个ORF的C-末端融合有17种标签，包括可见的报告基因和表位标签，并且可以通过4种抗性标记进行选择。我们通过观察与细胞器靶向信号融合并由气孔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R4 DS Gateway克隆系统在拟南芥中功能良好。我们还证实了R4 DS Gateway克隆系统中的两个克隆位点是等同的，并且是独立调控的。所获得的结果表明，R4 DS网关克隆系统有利于详细比较两个启动子的表达模式，以及在植物中的特定细胞中的两个蛋白质的共定位和相互作用分析。
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.