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

• Published in: PloS one Vol. 12; no. 5; p. e0177889
• Main Authors: Aboulela, Mostafa, Tanaka, Yuji, Nishimura, Kohji, Mano, Shoji, Nishimura, Mikio, Ishiguro, Sumie, Kimura, Tetsuya, Nakagawa, Tsuyoshi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16.05.2017; Public Library of Science (PLoS)
• Subjects: Agronomy; Analysis; Arabidopsis - genetics; Arabidopsis thaliana; Assembling; Assembly; Asymmetry; Binary systems (materials); Biochemistry; Biodiversity; Bioinformatics; Biological evolution; Biology; Biology and Life Sciences; Botany; Cassettes; Cell division; Chloroplasts; Cloning; Cloning vectors; Cloning, Molecular - methods; Compatibility; Construction; Cotyledons; Deoxyribonucleic acid; Differentiation; DNA; Endoglucanase; Epitopes; F1-ATPase; Flexibility; Fluorescence; Fragmentation; Gene expression; Genetic crosses; Genetic transformation; Genetic Vectors - genetics; Genetically modified plants; Genomes; Genomics; Glyoxysomes; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Greening; Interdisciplinary aspects; Laboratories; Markers; Mathematical analysis; Methods; Microbiology; Molecular interactions; Nucleotide sequence; Offspring; Open Reading Frames; Peroxisomes; Plant Proteins - genetics; Plant Proteins - metabolism; Position (location); Promoter Regions, Genetic; Promoters; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombination; Research and Analysis Methods; Researchers; Science; Stomata; Technology; Tobacco; Tomatoes; Transcription factors; Transformation; Transgenic plants; Japan
• DOI: 10.1371/journal.pone.0177889
• ISSN: 1932-6203

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.