Abstract
BackgroundRecent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.ResultsHere we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations.ConclusionThis modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.
Highlights
Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously
A total of twelve modified pENTR vectors have been generated, comprising six different tags for both N- and C-terminal fusions, and we show that these constructs can be used to study proteins of interest following introduction of these vectors into Arabidopsis or following transient Agrobacterium-mediated transformation and co-transformation in Nicotiana benthamiana leaves [8]
Enhanced Yellow Fluorescent Protein (EYFP) and Enhanced Cyan Fluorescent Protein (ECFP) were chosen for localization analyses and for in vivo protein-protein interaction studies by FRET, HA and cMyc were selected as epitope tags for immunoprecipitation, and GST and STREP tags were chosen for affinity purification assays
Summary
Recent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale. The flood of information resulting from the sequencing of a number of plant genomes and other recent developments have led to a huge increase in the number of studies carried out using plants transformed with DNA fragments to overexpress or modulate the expression of a gene of interest. While the process of Agrobacterium tumefaciensmediated plant transformation has improved over the last (page number not for citation purposes)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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 call
