Optimizing plant transporter expression in Xenopus oocytes
© Feng et al.; licensee BioMed Central Ltd. 2013
Received: 2 October 2013
Accepted: 13 December 2013
Published: 20 December 2013
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. 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.
We 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.
The 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.
Heterologous expression systems are often used for the functional characterization of a gene. Xenopus laevis oocytes are widely used to express membrane proteins and channels. Over twenty years ago, the first plant membrane proteins were expressed in oocytes and these were a hexose transporter and a K+ channel [1, 2]. Since then, many plant membrane proteins including carriers [3–5], channels [6–9] and aquaporins [10–13] have been successfully expressed in oocytes. Oocyte expression was used to demonstrate function for the first plant nitrate transporter (Chl1, AtNRT1.1 or AtNPF6.3) that was identified and later for many more family members [3, 14–20]. 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, 21–23]. The high affinity rice nitrate transporter, OsNRT2.3a needs a partner protein, OsNAR2.1 for function in oocytes [22, 23].
Although all organisms generally share the same genetic code, each genus has evolved a slightly different pattern of codon usage. Heterologous protein expression in a foreign host may be diminished by factors such as biased codon usage, GC content and repeat sequences. To overcome these limitations, codon optimization can be used to enhance gene expression in various host cells. Heterologous synthetic genes with codon optimization showed increased expression levels in various organisms including E. coli[24, 25], yeast  and mammalian cells [27, 28]. For many plant transporters expressed in Xenopus oocytes, the low levels of expression can often limit the functional assay, making the detailed characterization of the protein difficult. In the past, it was speculated that differing codon bias may explain the very low levels of expression of some plant proteins in oocytes . Improvements in DNA synthesis technology have enabled the technique to be used for cost-effective gene cloning. Commercial suppliers make it possible to obtain the synthetic DNA with codon optimization in just a few weeks. In this study, DNA of the rice genes OsNAR2.1 and OsNRT2.3a were codon optimized and synthesized for oocyte expression. The cRNA of OsNAR2.1 and OsNRT2.3a were then synthesized using a commercially available kit. We compared how this process may improve the functional activity of plant nitrate transporter proteins expressed in oocytes. The nitrate transport activity was assayed using 15N-enriched nitrate uptake and the two-electrode voltage clamp technique.
Results and discussion
Codon optimization of OsNAR2.1 and OsNRT2.3a
DNA sequence parameters of optimized plant transporter genes OsNAR2.1 and OsNRT2.3a
GC content (%)
Max direct repeat
Max inverted repeat
Max dyad repeat
363, 540, 73
Nitrate uptake of oocytes
Electrophysiological analyses of oocytes
Enhanced transporter activity in oocytes
The expression of foreign proteins in oocytes has long been known to be improved by the inclusion of a polyA tail and the use of expression vectors that include Xenopus globin flanking UTR (untranslated region) sequence alongside the foreign DNA [1, 29, 30]. The polyA tail is recognized to improve mRNA stability and lifetime in the oocyte thereby improving the production of a foreign protein. The frog globin flanking UTR sequence is thought to make the heterologous cDNA more Xenopus-like and therefore thought to improve translation [29, 30]. Similarly the mRNA produced from the synthetic DNA is more like Xenopus message and this has resulted in improved translational efficiency in the oocyte. For OsNAR2.1 the removal of some repeat sequence (Table 1) may also have given better translation of the foreign protein. Making the GC content more like the 50% found in Xenopus  is likely to improve expression of plant genes. In Arabidopsis the GC content was reported as 44%, on the other hand in maize, rice and barley the figure was higher at >60% . Mammals usually have around 44% GC in their coding sequence and experimental work directly comparing low-GC genes with their high-GC counterparts showed 100-fold greater expression in the GC-rich genes . This study also showed that the mRNA degradation rate was independent of the GC content.
Together these data clearly show the methodological advantage for plant genes of using a synthetic template that has codon usage more like that found in Xenopus. It is widely accepted that optimized codons help to achieve faster translation rates and high accuracy in bacteria, yeast and mammalian cells [24–28, 34] and we now show this has advantages for plant genes in Xenopus oocytes too.
In this study, rice nitrate transporter OsNAR2.1 and OsNRT2.3a were codon optimized for Xenopus laevis. Nitrate transport activity was analyzed and compared between oocytes injected with different sources of template DNA. The optimization changes the DNA, but not the protein sequence. Compared with the original plant genes, oocytes injected with optimized genes had increased nitrate uptake and larger currents in electrophysiological analyses suggesting that there was an increased level of protein expression. Taken together, these data show that the codon optimized template can give much improved expression and therefore provides a big advantage when aiming to functionally characterize a plant transporter protein in the Xenopus oocyte system. Although this may not be the case for all plant transporter genes the relatively cheap cost of DNA synthesis now makes this worthwhile when using oocyte expression.
Cloning and mRNA synthesis of OsNAR2.1 and OsNRT2.3a
OsNAR2.1 and OsNRT2.3a were codon optimized and synthesized by the Genescript Company. cDNAs were then subcloned into the BglII and SpeI sites of the oocyte expression vector pT7TS  using a directional cloning method. Original OsNAR2.1 and OsNRT2.3a construct are as described previously . Plasmid was linearized by BamH I (Roche) and purified by PCR purification kit (QIAGEN). The mRNA synthesis kit (mMESSAGE mMACHINE® T7 Kit, Ambion) was used to synthesized the mRNA of all genes. A compressed air system (Harvard) was used for injection . Glass tips were calibrated for injection using known volumes and 1 μl mRNA mixtures (0.5 μg OsNAR2.1 and 1 μg OsNRT2.3a in total) were used to inject around 22–25 oocytes. Thus mRNA mixtures were injected as 25 ng: 50 ng, OsNAR2.1: OsNRT2.3a, and this weight ratio was chosen to reflect the differing molecular sizes and give similar molecular ratios [22, 23]. Oocytes from three different frogs were used for the data shown.
NO3- accumulation and 15N-NO3- uptake in oocytes
After injection, the oocytes were incubated for 2 days in NO3- free MBS solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.71 mM CaCl2, 0.82 mM MgSO4 and 15 mM HEPES, pH 7.4). The solution contains 10 g/ml sodium penicillin and 10 g/ml streptomycin sulphate. For NO3- measurement, oocytes were incubated in MBS solution containing 0.5 mM NaNO3 at 18°C for 16 h. After incubation, oocytes were washed with NO3- free MBS for four times. Four oocytes were collected as one sample in 1.5 mL tube, and 100 μl H2O was added into the tube. Lyses the oocytes and centrifuge the tube at 13000 rpm. The supernatants (40 μl) were collected for NO3- assay using the kit (Nitrate/Nitrite Colorimetric Assay kit, Cayman).
For 15N-NO3- measurement, oocytes were incubated in MBS solution containing 0.5 mM Na15NO3 with a 99% atom excess of 15N for 8 h and 16 h. oocytes were washed four times with ice-cold 0.5 mM NaNO3 MBS. Single oocyte was transferred to an empty tin capsule and then dried at 60°C for one week. Analysis for total 15N content using a continuous-flow isotope ratio mass spectrometer coupled with a carbon nitrogen elemental analyzer (ANCA-GSLMS; PDZ Europa). The delta-15N was calculated as described previously .
Two-electrode voltage clamp analysis
The nitrate-elicited currents were recorded in oocyte using two-electrode voltage clamp method (pClamp 10.2, Axon). The oocytes were incubated in nitrate-free MBS and then treated with MBS containing 0.5 mM sodium nitrate. Membrane potential of oocytes was pulsed from 0 to -160 mV with 20 mV incremental steps. The currents were recorded to obtain current–voltage curves [2–4].
This work was funded by the China 973 Program (grant number 2011CB100300), the National Natural Science Foundation, the PAPD and 111 Project (grant number 12009). AJM is supported by grant funding BB/JJ004553/1 from the BBSRC and the John Innes Foundation. The oocytes used in this work were kindly donated by the Gurdon Institute, Cambridge, UK.
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