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.

Mostafa Aboulela,Sumie Ishiguro,Kohji Nishimura,Yuji Tanaka,Mikio Nishimura,Shoji Mano,Tetsuya Kimura,Tsuyoshi Nakagawa,Carl Ng

doi:10.1371/journal.pone.0177889

Abstract

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.

Highlights

With advances in omics databases and bioinformatics, many genes and their products have been anticipated to work cooperatively
We developed the R4 dual-site (R4DS) Gateway cloning system, which permits the easy and fast cloning of two sets of promoters and open reading frames (ORFs) in a binary vector for plant transformation using any combination of a promoter entry clone and ORF entry clone
ORFs may be fused with various tags, if necessary

Summary

Introduction

With advances in omics databases and bioinformatics, many genes and their products have been anticipated to work cooperatively. In order to test this system, we made two-gene constructs with a combination of stomatal lineage-specific promoters and organelle-targeted fluorescent proteins, and observed consistent expression and localization patterns in transgenic Arabidopsis thaliana.

Results

Conclusion