Ferdowsi University of Mashhad

Document Type : Research Articles

Authors

1 Ferdowsi University of Mashhad

2 Department of Biotechnology and plant breeding, Ferdowsi University of Mashhad, Mashhad, Iran

3 Razi Vaccine and Serum Research Institute, Ministry of Agriculture, Mashhad, Iran

4 Horticulture research Institute, Shahid Bahonar University, Kerman, Iran

Abstract

An Agrobacterium-mediated transient gene expression assay was carried out in alfalfa (Medicago sativa) leaves for expression of a chimeric gene encoding a part of capsid protein of Foot and Mouth Disease virus called VP1. The plant leaves were transformed via agroinfiltration procedure. The presence of the foreign gene and its expression in transformed plants were evaluated by polymerase chain reaction (PCR), real time PCR, protein Dot blot and ELISA. Moreover, gene expression in the transformed leaves was quantified by ELISA method. The results obtained in this investigation indicated high level of gene expression in alfalfa leaves, showing that transient gene expression can be applied as an effective and time-saving procedure for the production of recombinant proteins. The procedures for transformation, detection of recombinant protein and its application for molecular experiments are described in the study.

Keywords

1. Bachrach H. L., Moore D. M., Mckercher P. D. Polatnick J. (1975) Immune and antibody response to an isolated capsid protein of foot and mouth disease virus. Journal of Immunology 115: 1636-1641
2. Bhat. R., Lahaye. T. and Panstruga. R. (2006) The visible touch: in planta visualization of protein-protein interactions by fluorophorebased methods. Plant Methods 2: 12-26.
3. Carter J. E. and Langridge W. H. R. (2002) Plant-based vaccines for protection against infectious and autoimmune diseases. Critical Reviews in Plant Sciences 21:93–109.
4. Doran P. M. (1999) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:426− 432
5. Fischer R. and Schillberg S. (2004) Molecular Farming. WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim. pp 329
6. Habibi M. and Zibaee S. (2013) Plant-based recombinant vaccines. International Journal of Agriculture and Crop Sciences 6: 27-30
7. Habibi-Pirkoohi M., Malekzadeh-Shafaroudi S., Marashi H., Moshtaghi N., Nasiri M. and Zibaee S. (2014) Transient expression of foot and mouth disease virus (FMDV) coat protein in Tobacco (Nicotiana tabacom) via Agroinfiltration. Iranian Journal of Biotechnology 12(3): 1015-1021
8. Ihara Y., Nagano M., Muto S., Uchimiya H. and Kawai M. (2007) Cell death suppressor Arabidopsis Bax inhibitor- 1 is associated with calmodulin binding and ion homeostasis. Plant Physiology 143: 650–660.
9. Janssen B. and Gardner R. (1989) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Molecular Biology 14:61–72.
10. Koprowski H. and Yusibov V. (2001) The green revolution: plants as heterologous expression vectors. Vaccine 19: 2735–41
11. Leckie B. M. and Stewart C. N. (2011) Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants. Plant Cell Reports 30: 325–334.
12. Rodriguez L. L. and Grubman M. J. (2009) Foot and mouth disease virus vaccines. Vaccine 27 (4): 90-94.
13. Sala F., Rigano M., Barbante A., Basso B., Walmsley A. M. and Castiglione S. (2003) Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives. Vaccine 21:803–808.
14. Santos-Rosa M. A., Poutaraud D., Merdinoglu D. and Mestre P. (2008) Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Reports 27:1053-1063.
15. Sohn S., Huh S. M., Kim K. H., Park J. W. and Lomonossoff G. (2011) Effect of rice stripe virus NS3 on transient gene expression and transgene co-silencing. Plant Pathology Journal 27(4): 310-314.
16. Sparkes IA., Runions J., Kearns A. and Hawes C. (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature Protocols 1(4): 2019-25.
17. Tang X., Frederick R. D., Zhou J., Halterman D. A., Jia Y. and Martin GB. (1996) Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 274: 2060–2063.
18. Vaquero C., Sack M., Chandler J., Jürgen D., Schuster F., Monecke M., Schillberg S. and Fischer R. (1999) Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves. PNAS, Proceedings of the National Academy of Sciences 96:11128–11133.
19. Walmsley A. M. and Arntzen C. J. (2000) Plants for delivery of edible vaccines. Current Opinion in Biotechnology 11:126–9.
20. Wang C. Y., Chang T. Y., Walfield A. M., Ye J. and Shen M. (2002) Effective synthetic peptide vaccine for foot-and-mouth disease in swine. Vaccine 20: 2603–2610.
21. Wroblewski T., Tomczak A. and Michelmore R. (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis Plant Biotechnology Journal 3: 259–273.
22. Yasmin A., Jalbani A., Ali M., Nasreen A. and Debener T. (2013) Development of Agrobacterium-based transient gene expression assay in rose leaves. Pakistan Journal of Botany 45 (3): 1005-1009
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