Abstract
A major obstacle in the genetic manipulation of tomato mosaic virus (ToMV) is the instability of the plasmid containing the infectious full-length cDNA of the ToMV vector, which often prevents the subcloning of a foreign gene of interest into the vector. We found that an insertion of a 0.3–1.6-kbp DNA fragment in the movement protein (MP) coding region effectively attenuated bacterial toxicity of the plasmid and greatly increased plasmid yield. Accumulation of a modified ToMV containing a 0.3-kb insertion in the MP coding region was comparable to that of a modified ToMV without an insertion in tobacco BY-2 protoplasts, while an insertion more than 0.6 kb significantly reduced accumulation of the viral RNA. The modified ToMV vector containing a 0.3-kb insertion was easily manipulated to introduce a coding sequence for human interferon-gamma (HuIFN-γ) and successfully utilized to produce HuIFN-γ in both BY-2 protoplasts and transgenic BY-2 cells.
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Acknowledgments
We thank Dr. Nam-Hai Chua for plasmid, and Mses. Sayuri Hamada, Akiko Mizuno, Akiko Taki and Chizuko Kaneko, and Messrs. Tomoaki Satoh and Shin-ya Kunikado for technical assistance. This work was supported by a grant from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency. Analysis of DNA sequencing was conducted with CREST-Akita Plant Molecular Science Satellite Laboratory.
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Dohi, K., Tamai, A. & Mori, M. Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector. Arch Virol 153, 1667–1675 (2008). https://doi.org/10.1007/s00705-008-0165-z
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DOI: https://doi.org/10.1007/s00705-008-0165-z