Simultaneous production of two foreign proteins from a polyvirus-based vector

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertio...

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Bibliographic Details
Published in:Virus research 2005-09, Vol.112 (1-2), p.1-8
Main Authors: Beauchemin, Chantal, Bougie, Véronique, Laliberté, Jean-François
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Base Sequence
Brassica - virology
Genetic Engineering - methods
Genetic Vectors
Glucuronidase - genetics
Glucuronidase - metabolism
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Molecular Sequence Data
Plant Diseases - virology
Polyproteins - chemistry
Polyproteins - genetics
Potyvirus - genetics
Potyvirus - metabolism
Transfection
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Summary:With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.
ISSN:0168-1702