A modified Gateway cloning strategy for overexpressing tagged proteins in plants.

Plant Methods
Manu J DubinGiovanna Benvenuto

Abstract

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. Here 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 e...Continue Reading

References

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Citations

Dec 7, 2013·BMC Plant Biology·Xu WangYong-Fu Fu
Jan 31, 2012·Plant Science : an International Journal of Experimental Plant Biology·Erin EgelkroutJohn A Howard
Feb 21, 2009·Plant Biotechnology Journal·Leandro Hueso EstornellAntonio Granell
Feb 20, 2014·The Plant Journal : for Cell and Molecular Biology·Laia ArmengotMaría Carmen Martínez
Jul 29, 2010·The Biochemical Journal·Jens Tilsner, Karl J Oparka
May 31, 2015·Plant Science : an International Journal of Experimental Plant Biology·Harin JungJu-Kon Kim
Dec 28, 2016·International Journal of Molecular Sciences·Min Kyoung YouSun-Hwa Ha
Oct 25, 2011·Nature·Daniel J GibbsMichael J Holdsworth
Apr 12, 2016·Physiologia Plantarum·Mingjie Chen, Jay J Thelen
Feb 9, 2020·International Journal of Molecular Sciences·Sangkyu ParkSun-Hyung Lim
Dec 20, 2013·Plant Physiology·Fernanda SalvatoIan Max Møller
May 1, 2009·Plant Physiology·Songbiao ChenGuo-Liang Wang
Mar 3, 2010·Cold Spring Harbor Protocols·Manu J DubinGiovanna Benvenuto

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Datasets Mentioned

BETA
EU334816
EU334817
EU334818
EU334819
EU334820
EU334821
EU334822
EU334823
EU334824
EU334825

Methods Mentioned

BETA
Destination
affinity purification
PCR
co-immunoprecipitation
pull down
Immunoprecipitation
transgenic

Software Mentioned

MultiSite Gateway
MultiSite

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