Abstract
BackgroundWe have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. We utilized the full-length cDNA databases to generate a normalized library consisting of 239 full-length Arabidopsis thaliana transporter cDNAs. The genes were arranged into a 96-well format and optimized for expression in Xenopus oocytes by cloning each coding sequence into a Xenopus expression vector.ResultsInjection of 96 in vitro transcribed cRNAs from the library in pools of columns and rows into oocytes and subsequent screening for glucose uptake activity identified three glucose transporters. One of these, AtSTP13, had not previously been experimentally characterized.ConclusionExpression of the library in Xenopus oocytes, combined with uptake assays, has great potential in assignment of plant transporter function and for identifying membrane transporters for the many plant metabolites where a transporter has not yet been identified.
Highlights
We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function
We have developed a functional genomics approach to screen for function of plant transport proteins based on the classical expression cloning in Xenopus oocytes
Development of transporter cDNA library The Arabidopsis Membrane Protein Library (AMPL) includes all predicted polytopic Arabidopsis genes grouped into families based on sequence homology [4]
Summary
We have developed a functional genomics approach based on expression cloning in Xenopus oocytes to identify plant transporter function. With the completion of the Arabidopsis genome which presents a complete catalogue of all genes that may be expressed in the plant, it became apparent that 5–10% of the 25498 predicted proteins represent transport protein homologues [4]. Putative functions have been assigned to many of these proteins based on homology to characterized transporters in Arabidopsis and other organisms [5,6,7]. Phylogenetic relationships are useful for predicting structural and mechanistic properties, experimental evidence is essential for assigning function. This remains a major challenge for the majority of predicted transporter proteins in Arabidopsis
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.
