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Plant Methods
2013 Dec 20;91:48. doi: 10.1186/1746-4811-9-48.
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Optimizing plant transporter expression in Xenopus oocytes.
Feng H
,
Xia X
,
Fan X
,
Xu G
,
Miller AJ
.
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BACKGROUND: 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.
RESULTS: 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.
CONCLUSION: 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.
Figure 1. Alignment of synthetic and original OsNAR2.1 and OsNRT2.3a open reading frame sequences highlighting the base changes. In Table 1 more specific sequence differences are listed.
Figure 2. Nitrate accumulation in Xenopus oocytes. Mixed mRNA of either synthetic genes (synthetic OsNAR2.1 and OsNRT2.3a) or the original genes (original OsNAR2.1 and OsNRT2.3a) were injected into oocytes. Oocytes were incubated in MBS with 0.5 mM NaNO3 for 16 h and washed four times with NO3- free MBS solution. Four oocytes were pooled for each sample. The values are means SE of four replicates with a and b indicating the statistical significance at p ≤ 0.05. The example shown is representative of the results from two frogs.
Figure 3. 15NO3-uptake in Xenopus oocytes. Mixed mRNA of either synthetic genes (synthetic OsNAR2.1 and OsNRT2.3a) or the original genes (original OsNAR2.1 and OsNRT2.3a) were injected into oocytes. Single oocyte was incubated in MBS with 0.5 mM Na15NO3 for 8 h and 16 h, and then washed four times with cold 0.5 mM NaNO3 before 15N analysis. Data are averaged and SE of five oocytes. a, b and c indicate the significant difference at p ≤ 0.05 of same template DNA between different treatments at 5% of probability. The example shown is representative of the results from two frogs.
Figure 4. Data spread analysis of15N-nitrate influx for individual oocytes injected with water or RNA. Injected oocytes were incubated in MBS solution containing 0.5 mM Na15NO3 for 8 and 16 h. Delta 15N influx of individual oocytes injected with water (blue), RNA prepared from synthetic template DNA (red) and original genes (green) were compared. The data are from 10-15 cells after 8 h (A) or 16 h (B) incubation. The line (orange) represents the expected 15N influx value for an oocyte injected with the original plant DNA-template RNA. At both 8 h (A) and 16 h (B), 100% of the synthetic-template RNA injected oocytes were above this threshold line, while the equivalent figure for the original plant DNA were 14 and 17% at 8 and 16 h respectively.
Figure 5. Current–voltage difference curves for oocytes expressing the rice nitrate transporter OsNAR2.1/OsNRT2.3a. These curves were recorded from oocytes injected with water (blue), synthetic (red) and original (green) genes. All the oocytes treated with 0.5 mM nitrate at pH 7.4. Results were average and SE values and obtained in three different oocytes from the same frog.
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