Transient expression of green fluorescent protein in radish (Raphanus sativus) using a turnip mosaic virus based vector

Document Type : Research paper

Authors

Department of Biotechnology, Institute of Science and High Technology and Environmental Sciences, Graduate University of Advanced Technology, P. O. Box: 76315-117, Kerman, Iran

Abstract

It is possible to use transgenic plants, as bioreactors, for the production of recombinant inexpensive chemicals and drug components. Transient gene expression is an appropriate alternative to stable transformation because it allows an inexpensive and rapid method for expression of recombinant proteins in plant tissues. In recent years, plant viral vectors have been increasingly developed as successful biotechnological tools for the expression of a wide range of foreign proteins in plants. Plant viruses-based vectors are useful because of the autonomous replication and the high level of gene expression in a short time. Here, we have used a vector derived from an infectious turnip mosaic virus (TuMV) for transient expression of the jellyfish green fluorescent protein (GFP), as a model heterologous protein, in radish plant. The GFP ORF was inserted between NIb and CP sites under control of CAMV35S promoter. The leaves were inoculated using surface scratch by carborundum and harvested 14 days after inoculation for analysis. The visualization of GFP fluorescence in leaf disks from inoculated plants using fluorescence microscopy demonstrated gene transformation and systemic infection. Expression of the desired protein were confirmed by RT-PCR, SDS-PAGE and Dot blotting analysis. The quantitative values of GFP in different inoculated leaves were compared by ELISA assay using an anti-GFP antibody. The results showed high level of expression of GFP protein in leaves of inoculated plants compared with wild type. The results demonstrated that the TuMV-based vector has high efficiency for the expression of the foreign protein in the radish plant. This is the first examination of TuMV-based vector in radish.

Keywords


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