Optimizing plant transporter expression in Xenopus oocytes

Plant Methods

Anthony J Miller²  Guohua Xu¹  Xiaorong Fan²  Xiudong Xia¹  Huimin Feng² 

[1] State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China;Department of Metabolic Biology, John Innes Centre, Norwich Research Park NR4 7UH, UK

关键词: Electrophysiology;    Uptake;    Nitrate transporter;    Plant;    Xenopus oocyte;    DNA optimization;   

Others  :  820159
DOI  :  10.1186/1746-4811-9-48

 received in 2013-10-02, accepted in 2013-12-13,  发布年份 2013

【 摘 要 】

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/ webcite; http://genomes.urv.es/OPTIMIZER/ webcite) 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.

【 授权许可】

   
2013 Feng et al.; licensee BioMed Central Ltd.

【 图 表 】

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