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
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.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
