Abstract
BackgroundRapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.ResultsWe used the generally available software (http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/) to predict a DNA sequence for the plant gene that is better suited for Xenopus laevis. Rice OsNAR2.1 and OsNRT2.3a DNA optimized sequences were commercially synthesized for Xenopus expression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and original OsNAR2.1 and OsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3- than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.ConclusionThe functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in the Xenopus oocyte system.
Highlights
Rapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins
Codon optimization of OsNAR2.1 and OsNRT2.3a There are some general rules that emerge from the analysis of the preferred codons in Xenopus and these can be used to optimize a gene sequence for expression in oocytes [30]
Using the codon usage bias software for Xenopus, the DNA gene sequence was optimized for OsNAR2.1 (LOC_Os02g38230) and OsNRT2.3a (LOC_Os01g50820) and the resulting DNA sequences were synthesized by the Genescript Company and named syn-OsNAR2.1 and syn-OsNRT2.3a
Summary
Rapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes. Xenopus laevis oocytes are widely used to express membrane proteins and channels. The first plant membrane proteins were expressed in oocytes and these were a hexose transporter and a K+ channel [1,2]. Oocyte expression was used to demonstrate function for the first plant nitrate transporter Some of the plant NRT2 nitrate transporter family members require a second gene NAR2 for function and this requirement was demonstrated using oocyte expression [4,5,21,22,23]. The high affinity rice nitrate transporter, OsNRT2.3a needs a partner protein, OsNAR2.1 for function in oocytes [22,23]
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