Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Plant Methods
2006 Oct 27;2:17. doi: 10.1186/1746-4811-2-17.
Show Gene links
Show Anatomy links
Screening for plant transporter function by expressing a normalized Arabidopsis full-length cDNA library in Xenopus oocytes.
Nour-Eldin HH
,
Nørholm MH
,
Halkier BA
.
???displayArticle.abstract???
We 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. Injection 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. Expression 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.
Figure 1. Screening for glucose uptake activity in Xenopus oocytes expressing pools of cRNA of Arabidopsis transporter cDNAs. cRNA of 96 genes transcribed with native plant UTRs were pooled in columns and rows and screened for glucose uptake in Xenopus oocytes. High glucose uptake was measured in column 5 and row C, which suggests that the marked well contains a glucose transporter.
Figure 2. Confirmation of glucose uptake activity by AtSTP13. cRNA of AtSTP13 representing the single clone C5 was injected into nine oocytes. Measurement of glucose uptake by the oocytes was compared to oocytes injected with row C and column 5, respectively.
Figure 3. Effect of different UTRs on gene expression in Xenopus oocytes. CDSs of AtSTP1, AtSTP4, AtSTP13, AtSUCI, and AtSUC2 were expressed with their native plant UTRs or with Xenopus-specific β-globin gene UTRs. Water injected oocytes functioned as negative controls for endogenous glucose or sucrose uptake by oocytes. Assays were performed with 15 μM radio labelled glucose or sucrose (~100 μCi/μmol).
Figure 4. Screening for glucose uptake in oocytes expressingpools of cRNA of Arabidopsis transporter cDNAs optimized for Xenopus expression. cRNAs of 96 genes transcribed with Xenopus UTRs were pooled in columns and rows and screened for glucose uptake in oocytes. High glucose uptake was measured in row A, C and E and column 4, 5 and 7, which suggests that the marked wells, contain glucose transporters.
Arabidopsis Genome Initiative,
Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.
2000, Pubmed
Arabidopsis Genome Initiative,
Analysis of the genome sequence of the flowering plant Arabidopsis thaliana.
2000,
Pubmed
Boorer,
Functional expression of a plant plasma membrane transporter in Xenopus oocytes.
1992,
Pubmed
,
Xenbase
Büttner,
Monosaccharide transporters in plants: structure, function and physiology.
2000,
Pubmed
Cao,
Expression of an outward-rectifying potassium channel from maize mRNA and complementary RNA in Xenopus oocytes.
1992,
Pubmed
,
Xenbase
Carninci,
Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.
2001,
Pubmed
Cestari,
A PCR shortcut to oocyte expression.
1993,
Pubmed
,
Xenbase
Chen,
Characterization of glucosinolate uptake by leaf protoplasts of Brassica napus.
2000,
Pubmed
Chen,
Long-distance phloem transport of glucosinolates in Arabidopsis.
2001,
Pubmed
Dawson,
Estimated Contributions of Root and Shoot to the Nicotine Content of the Tobacco Plant.
1959,
Pubmed
Krieg,
Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs.
1984,
Pubmed
,
Xenbase
Liu,
The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes.
1998,
Pubmed
MacAulay,
Water transport by the human Na+-coupled glutamate cotransporter expressed in Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Miller,
Xenopus oocytes as an expression system for plant transporters.
2000,
Pubmed
,
Xenbase
Norholm,
Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.
2006,
Pubmed
,
Xenbase
Nour-Eldin,
Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments.
2006,
Pubmed
Romero,
Expression cloning using Xenopus laevis oocytes.
1998,
Pubmed
,
Xenbase
Saier,
Tracing pathways of transport protein evolution.
2003,
Pubmed
Sauer,
SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein.
1994,
Pubmed
Seki,
Functional annotation of a full-length Arabidopsis cDNA collection.
2002,
Pubmed
Seki,
High-efficiency cloning of Arabidopsis full-length cDNA by biotinylated CAP trapper.
1998,
Pubmed
Sherson,
Monosaccharide/proton symporter AtSTP1 plays a major role in uptake and response of Arabidopsis seeds and seedlings to sugars.
2000,
Pubmed
Truernit,
The sink-specific and stress-regulated Arabidopsis STP4 gene: enhanced expression of a gene encoding a monosaccharide transporter by wounding, elicitors, and pathogen challenge.
1996,
Pubmed
Véry,
Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents.
1995,
Pubmed
,
Xenbase
Ward,
Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana.
2001,
Pubmed
Yamada,
Empirical analysis of transcriptional activity in the Arabidopsis genome.
2003,
Pubmed
Zhou,
Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine.
1998,
Pubmed
,
Xenbase