Molecular Biotechnology

, Volume 61, Issue 2, pp 84–92 | Cite as

The Effect of Methyl Jasmonate and Temperature on the Transient Expression of Recombinant Proteins in Cucurbita pepo L.

  • Vahid Karimzadegan
  • Vahid Jalali Javaran
  • Masoud Shams Bakhsh
  • Mokhtar Jalali JavaranEmail author
Original Paper


The aim of this study is to assess the effect of methyl jasmonate (MeJA) and temperature on the valuable pharmaceuticals expression in a virus-mediated transient expression system, and so the Zuchini Yellow Mosaic Virus (ZYMV) based vector was used for transferring the GFP reporter gene and recombinant tissue plasminogen activator (rtPA) gene (K2S) to cucurbit (Cucurbita pepo L.). MeJA, temperature and time (days after inoculation), were evaluated as a factorial experiment in a completely randomized design (CRD). At first, the effect of all treatment combinations on GFP expression was assessed. At this step, the ELISA test was used to select the optimum treatment combination. ELISA method revealed the significant difference between applied treatments. The optimized treatment significantly increased the expression of rtPA compared to the control. The Real-Time PCR reaction for both GFP and rtPA genes showed no significant differences between optimum and control treatments, however, transcripts of the small subunit of RuBisCO were extremely down-regulated in optimum treatment condition. Reduction in RuBisCO expression at protein level was tangible under treatment condition based on the ELISA test. Therefore, it can be inferred that suppressing the expression of RuBisCO, probably resulted in higher access of expression system to free amino acids inside the cell. In this study, MeJA has been shown to be a positive factor, but the low temperature (17 °C), unlike previous studies, suppressed the expression of recombinant protein unexpectedly, probably due to the incompatibility of the viral construct with low temperature. In conclusion, the use of a suitable gene construct, which is not sensitive to temperature, is likely to result in a more favorable outcome.


Recombinant protein Methyl Jasmonate Transient expression Elicitor Molecular Pharming 


  1. 1.
    Spiegel, H., Stöger, E., Twyman, R. M., & Buyel, J. F. (2018). Current status and perspectives of the molecular farming landscape. Molecular Pharming: Applications, Challenges, and Emerging Areas. Google Scholar
  2. 2.
    Fischer, R., Schillberg, S., Buyel, F., J., and Twyman, M., R (2013). Commercial aspects of pharmaceutical protein production in plants. Current Pharmaceutical Design, 19(31), 5471–5477.CrossRefGoogle Scholar
  3. 3.
    Gomord, V., Chamberlain, P., Jefferis, R., & Faye, L. (2005). Biopharmaceutical production in plants: Problems, solutions and opportunities. Trends. Biotechnol., 23(11), 559–565.CrossRefGoogle Scholar
  4. 4.
    Sainsbury, F., & Lomonossoff, G. P. (2014). Transient expressions of synthetic biology in plants. Current Opinion in Plant Biology, 19, 1–7.CrossRefGoogle Scholar
  5. 5.
    Weathers, P. J., Towler, M. J., & Xu, J. (2010). Bench to batch: advances in plant cell culture for producing useful products. Applied Microbiology and Biotechnology, 85(5), 1339–1351.CrossRefGoogle Scholar
  6. 6.
    Ellis, R. J. (1979). The most abundant protein in the world. Trends in Biochemical Science, 4(11), 241–244.CrossRefGoogle Scholar
  7. 7.
    Cavalcante, A. P. R., Jacinto, T., & Machado, O. L. T. (1999). Methyl jasmonate changes the levels of rubisco and other leaf proteins in Ricinus communis. Acta Physiology Plant, 21(2), 161–166.CrossRefGoogle Scholar
  8. 8.
    Attaran, E., Major, I. T., Cruz, J. A., Rosa, B. A., Koo, A. J., Chen, J., Kramer, D. M., He, S. Y., & Howe, G. A. (2014). Temporal dynamics of growth and photosynthesis suppression in response to jasmonate signaling. Plant Physiology, 165(3), 1302–1314.CrossRefGoogle Scholar
  9. 9.
    Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S. Y., & Howe, G. A. (2007). JAZ repressor proteins are targets of the SCF COI1 complex during jasmonate signalling. Nature, 448(7154), 661.CrossRefGoogle Scholar
  10. 10.
    Hou, X., Lee, L. Y. C., Xia, K., Yan, Y., & Yu, H. (2010). DELLAs modulate jasmonate signaling via competitive binding to JAZs. Development Cell, 19(6), 884–894.CrossRefGoogle Scholar
  11. 11.
    Chini, A., Fonseca, S., Fernandez, G., Adie, B., Chico, J. M., Lorenzo, O., Garcia-Casado, G., López-Vidriero, I., Lozano, F. M., Ponce, M. R., & Micol, J. L. (2007). The JAZ family of repressors is the missing link in jasmonate signalling. Nature., 448(7154), 666.CrossRefGoogle Scholar
  12. 12.
    Desai, P. N., Shrivastava, N., & Padh, H. (2010). Production of heterologous proteins in plants: Strategies for optimal expression. Biotechnology Advances, 28(4), 427–435.CrossRefGoogle Scholar
  13. 13.
    Kim, S. T., Cho, K. S., Yu, S., Kim, S. G., Hong, J. C., Han, C. D., Bae, D. W., Nam, M. H., & Kang, K. Y. (2003). Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells. Proteomics, 3(12), 2368–2378.CrossRefGoogle Scholar
  14. 14.
    Robert, S., Goulet, M. C., D’Aoust, M. A., Sainsbury, F., & Michaud, D. (2015). Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein. Plant Biotechnology Journal, 13(8), 1169–1179.CrossRefGoogle Scholar
  15. 15.
    Fraissinet-Tachet, L., Baltz, R., Chong, J., Kauffmann, S., Fritig, B., & Saindrenan, P. (1998). Two tobacco genes induced by infection, elicitor and salicylic acid encode glucosyltransferases acting on phenylpropanoids and benzoic acid derivatives, including salicylic acid. FEBS Letters, 437(3), 319–323.CrossRefGoogle Scholar
  16. 16.
    Okada, K., Abe, H., & Arimura, G. I. (2014). Jasmonates induce both defense responses and communication in monocotyledonous and dicotyledonous plants. Plant Cell Physiology, 56(1), 16–27.CrossRefGoogle Scholar
  17. 17.
    Faye, L., Boulaflous, A., Benchabane, M., Gomord, V., & Michaud, D. (2005). Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine, 23(15), 1770–1778.CrossRefGoogle Scholar
  18. 18.
    Doran, P. M. (2006). Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnology, 24(9), 426–432.CrossRefGoogle Scholar
  19. 19.
    De Neve, M., De Loose, M., Jacobs, A., Van Houdt, H., Kaluza, B., Weidle, U., Van Montagu, M., & Depicker, A. (1993). Assembly of an antibody and its derived antibody fragment in Nicotiana and Arabidopsis. Transgenic Research, 2(4), 227–237.CrossRefGoogle Scholar
  20. 20.
    Outchkourov, N. S., Rogelj, B., Strukelj, B., & Jongsma, M. A. (2003). Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production. Plant Physiology, 133(1), 379–390.CrossRefGoogle Scholar
  21. 21.
    Khoudi, H., Laberge, S., Ferullo, J. M., Bazin, R., Darveau, A., Castonguay, Y., Allard, G., Lemieux, R., & Vézina, L. P. (1999). Production of a diagnostic monoclonal antibody in perennial alfalfa plants. Biotechnology and Bioengineering, 64(2), 135–143.CrossRefGoogle Scholar
  22. 22.
    Sharp, J. M., & Doran, P. M. (2001). Characterization of monoclonal antibody fragments produced by plant cells. Biotechnology and Bioengineering, 73(5), 338–346.CrossRefGoogle Scholar
  23. 23.
    Hehle, V. K., Paul, M. J., Drake, P. M., Ma, J. K., & van Dolleweerd, C.J. (2011) Antibody degradation in tobacco plants: a predominantly apoplastic process. BMC Biotechnology. 11(1), 128.CrossRefGoogle Scholar
  24. 24.
    Weaver, L. M., Himelblau, E., & Amasino, R. M. (1997). Leaf senescence: gene expression and regulation, in Genetic engineering (pp. 215–234). Boston: Springer.Google Scholar
  25. 25.
    Smart, C. M. (1994). Gene expression during leaf senescence. New Phytology, 126(3), 419–448.CrossRefGoogle Scholar
  26. 26.
    Zhao, Y., Ge, W., Kong, Y., & Zhang, C. (2003). Cloning, expression, and renaturation studies of reteplase. Journal of Microbiology and Biotechnology, 13(6), 989–992.Google Scholar
  27. 27.
    Hafizi, A., Malboobi, M. A., Jalali-Javaran, M., Maliga, P., & Alizadeh, H. (2017). Covalent-display of an active chimeric-recombinant tissue plasminogen activator on polyhydroxybutyrate granules surface. Biotechnology Letters, 39(11), 1683–1688.CrossRefGoogle Scholar
  28. 28.
    Abdoli-Nasab, M., Jalali-Javaran, M., Cusidó, R. M., Palazón, J., Baghizadeh, A., & Alizadeh, H. (2013). Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast. Molecular Biology Reports, 40(10), 5749–5758.CrossRefGoogle Scholar
  29. 29.
    Javaran, V. J., Shafeinia, A., Javaran, M. J., Gojani, E. G., & Mirzaee, M. (2017). Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector. Biotechnology Letters, 39(4), 607–612.CrossRefGoogle Scholar
  30. 30.
    Hidalgo, D., Abdoli-Nasab, M., Jalali-Javaran, M., Bru-Martínez, R., Cusidó, R. M., Corchete, P., & Palazon, J. (2017). Biotechnological production of recombinant tissue plasminogen activator protein (reteplase) from transplastomic tobacco cell cultures. Plant Physiology and Biochemistry, 118, 130–137.CrossRefGoogle Scholar
  31. 31.
    Hsu, C. H., Lin, S. S., Liu, F. L., Su, W. C., & Yeh, S. D. (2004). Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis. The Journal of Allergy and Clinical Immunology, 113(6), 1079–1085.CrossRefGoogle Scholar
  32. 32.
    Obrero, A., Die, J. V., Román, B., Gómez, P., Nadal, S., & González-Verdejo, C. I. (2011). Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR. Journal of Agriculture and Food Chemistry, 59(10), 5402–5411.CrossRefGoogle Scholar
  33. 33.
    Goossens, A., Van Montagu, M., & Angenon, G. (1999). Co-introduction of an antisense gene for an endogenous seed storage protein can increase expression of a transgene in Arabidopsis thaliana seeds. FEBS Letters, 456(1), 160–164.CrossRefGoogle Scholar
  34. 34.
    Stanton, M. A., Ullmann-Zeunert, L., Wielsch, N., Bartram, S., Svatoš, A., Baldwin, I. T., & Groten, K. (2013). Silencing ribulose-1, 5-bisphosphate carboxylase/oxygenase expression does not disrupt nitrogen allocation to defense after simulated herbivory in Nicotiana attenuata. Plant Signal Behavavior, 8(12), 27570.CrossRefGoogle Scholar
  35. 35.
    Murby, M., Uhlén, M., & Ståhl, S. (1996). Upstream strategies to minimize proteolytic degradation upon recombinant production in Escherichia coli. Protein Expression and Purification, 7(2), 129–136.CrossRefGoogle Scholar
  36. 36.
    Stevens, L. H., Stoopen, G. M., Elbers, I. J., Molthoff, J. W., Bakker, H. A., Lommen, A., … and Jordi, W. (2000). Effect of climate conditions and plant developmental stage on the stability of antibodies expressed in transgenic tobacco. Plant Physiology, 124(1), 173–182.CrossRefGoogle Scholar
  37. 37.
    Kosaka, Y., Ryang, B. S., Kobori, T., Shiomi, H., Yasuhara, H., & Kataoka, M. (2006). Effectiveness of an attenuated Zucchini yellow mosaic virus isolate for cross-protecting cucumber. Plant Disease, 90(1), 67–72.CrossRefGoogle Scholar
  38. 38.
    Mason, H. S., DeWald, D. B., & Mullet, J. E. (1993). Identification of a methyl jasmonate-responsive domain in the soybean vspB promoter. The Plant Cell, 5(3), 241–251.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Vahid Karimzadegan
    • 1
  • Vahid Jalali Javaran
    • 2
  • Masoud Shams Bakhsh
    • 1
  • Mokhtar Jalali Javaran
    • 1
    Email author
  1. 1.Department of Biotechnology, Faculty of AgricultureTarbiat Modares UniversityTehranIran
  2. 2.Genetic Recourse Management Plan, Department of Biotechnology, Faculty of AgricultureTarbiat Modares UniversityTehranIran

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