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Optimizing plant transporter expression in Xenopus oocytes.

Huimin Feng, Xiudong Xia, Xiaorong Fan1

  • 1State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China. xiaorongfan@njau.edu.cn.

Plant Methods
|December 24, 2013
PubMed
Summary
This summary is machine-generated.

DNA codon optimization significantly enhanced the functional activity of a rice nitrate transporter in Xenopus oocytes. This method improves plant protein expression and characterization in heterologous systems.

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Biochemistry

Background:

  • Advances in DNA synthesis enable rapid gene cloning and protein characterization.
  • Xenopus laevis oocytes are a common system for heterologous protein expression, especially membrane proteins.
  • Low expression levels of plant transporters in oocytes hinder functional characterization.

Purpose of the Study:

  • To investigate if DNA codon optimization can enhance the functional activity of plant nitrate transporters in Xenopus oocytes.
  • To assess the efficiency of codon optimization for expressing plant proteins in a heterologous system.

Main Methods:

  • Utilized publicly available software to predict codon-optimized DNA sequences for rice nitrate transporter genes (OsNAR2.1 and OsNRT2.3a).
  • Commercially synthesized optimized DNA sequences and produced cRNA.
  • Injected cRNA into Xenopus laevis oocytes and performed nitrate uptake assays and two-electrode voltage clamp analysis.

Main Results:

  • Oocytes injected with codon-optimized cRNA showed significantly higher nitrate accumulation compared to those with original sequences.
  • Two-electrode voltage clamp analysis confirmed larger currents in oocytes expressing codon-optimized transporter genes.
  • Codon optimization led to improved expression and measurable functional activity of the rice nitrate transporter.

Conclusions:

  • DNA codon optimization effectively improved the functional activity of a rice high-affinity nitrate transporter in Xenopus oocytes.
  • This methodology provides a promising approach for enhancing the expression and functional characterization of plant proteins in oocyte systems.