State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China.
Plant Methods. 2013 Dec 20;9(1):48. doi: 10.1186/1746-4811-9-48.
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.
DNA 合成技术的快速进步正在彻底改变基因克隆和对其编码蛋白的表征。非洲爪蟾卵母细胞是表达和功能表征膜蛋白的常用异源系统。对于许多植物蛋白，特别是转运蛋白，低水平的表达可能会限制这些细胞中的功能活性，从而难以对蛋白质进行表征。合成 DNA 技术的改进现在使得优化植物 cDNA 在非洲爪蟾中的密码子用法变得快速、简单且相对便宜。我们已经测试了该优化过程是否可以提高在卵母细胞中测定的双组分植物硝酸盐转运体的功能活性。
我们使用了通用的软件（http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/）来预测更适合非洲爪蟾的植物基因的 DNA 序列。水稻 OsNAR2.1 和 OsNRT2.3a 的 DNA 优化序列已商业化用于非洲爪蟾表达。使用市售试剂盒用模板 DNA 合成 cRNA。将 cRNA 混合物注射到优化和原始 OsNAR2.1 和 OsNRT2.3a 的卵母细胞中。在 0.5mM Na15NO3 中孵育 16 小时后，用优化后的 DNA 模板合成的 cRNA 注射的卵母细胞比原始基因能积累更多的 NO3-。用优化后的模板合成的 cRNA 注射的卵母细胞的双电极电压钳分析证实，与原始水稻 cDNA 相比，优化后的模板产生了明显更大的电流。
通过对基因进行 DNA 密码子优化，提高了水稻高亲和力硝酸盐转运体在卵母细胞中的功能活性。该方法为提高植物蛋白在非洲爪蟾卵母细胞系统中的表达水平和更好地进行后续功能表征提供了前景。
