Skip to main content
Log in

Comparison of gfp Gene Expression Levels after Agrobacterium-Mediated Transient Transformation of Nicotiana rustica L. by Constructs with Different Promoter Sequences

  • Published:
Cytology and Genetics Aims and scope Submit manuscript

Abstract

Promoters are key elements regulating gene expression levels, therefore their selection is an important step in genetic engineering research. The reporter gene gfp, which encodes green fluorescent protein (GFP), was transiently expressed in leaf tissues of Aztec tobacco Nicotiana rustica L. Compared to other species of the Nicotiana genus, Aztec tobacco has a large potential for expression of heterologous proteins, a large vegetative biomass, can be easily infiltrated, and is unpretentious in cultivation. Six genetic constructs were used with different promoter sequences: the 35S promoter of Cauliflower Mosaic Virus (35S CaMV), the double-enhanced 35S promoter (D35S CaMV), promoters of the RbcS1B and RbcS2B genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) isolated from Arabidopsis thaliana (L.) Heynh., and promoters of the LHB1B1 and LHB1B2 genes from A. thaliana encoding chlorophyll a/b binding proteins. The gfp gene expression was detected visually, spectrofluorimetrically, and by protein content (Bradford assay) on the seventh day after infiltration. The highest level of expression was observed using the double-enhanced 35S promoter (D35S CaMV) and the lowest using the LHB1B1 gene promoter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

Similar content being viewed by others

REFERENCES

  1. Blazeck, J. and Alper, H., Systems metabolic engineering: genome-scale models and beyond, Biotechnol. J., 2010, vol. 5, no. 7, pp. 647–659. https://doi.org/10.1002/biot.200900247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Keasling, J.D., Manufacturing molecules through metabolic engineering, Science, 2010, vol. 330, no. 6009, pp. 1355–1358. https://doi.org/10.1126/science.1193990

    Article  CAS  PubMed  Google Scholar 

  3. Rosano, G.L. and Ceccarelli, E.A., Recombinant protein expression in Escherichia coli: advances and challenges, Front. Microbiol., 2014, vol. 5, no. 172, pp. 1–17.https://doi.org/10.3389/fmicb.2014.00172

    Article  Google Scholar 

  4. De Vooght, L., Caljon, G., Stijlemans, B., De Baetselier, P., Coosemans, M., and Van Den Abbeele, J., Expression and extracellular release of a functional anti-trypanosome Nanobody® in Sodalis glossinidius, a bacterial symbiont of the tsetse fly, Microb. Cell Fact., 2012, vol. 1, no. 11, p. 1–11. doi. org/https://doi.org/10.1186/1475-2859-11-23

    Article  CAS  Google Scholar 

  5. Sorensen, H.P. and Mortensen, K.K., Advanced genetic strategies for recombinant protein expression in Escherichia coli, J. Biotechnol., 2005, vol. 115, no. 2, pp. 113–128.https://doi.org/10.1016/j.jbiotec.2004.08.004

    Article  CAS  PubMed  Google Scholar 

  6. Orom, U.A., Nielsen, F.C., and Lund, A.H., MicroRNA-10a binds the 5’ UTR of ribosomal protein mRNAs and enhances their translation, Mol. Cell, 2008, vol. 30, no. 4, pp. 460–471. https://doi.org/10.1016/j.molcel.2008.05.001

    Article  CAS  PubMed  Google Scholar 

  7. Wilkie, G.S., Dickson, K.S., and Gray, N.K., Regulation of mRNA translation by 5'- and 3'-UTR-binding factors, Trends Biochem. Sci., 2003, vol. 28, no. 4, pp. 182–188. https://doi.org/10.1016/S0968-0004(03)00051-3

    Article  CAS  PubMed  Google Scholar 

  8. Leppek, K., Das, R., and Barna, M., Functional 5’ UTR mRNA structures in eukaryotic translation regulation and how to find them, Nat. Rev. Mol. Cell Biol., 2018, vol. 19, no. 3, pp. 158–174. https://doi.org/10.1038/nrm.2017.103

    Article  CAS  PubMed  Google Scholar 

  9. Becker, J., Wittmann, C., Advanced biotechnology: Metabolically engineered cells for the bio-based production of chemicals and fuels, materials and healthcare products, Angew. Chem. Int. Ed., 2015, vol. 54, no. 11, pp. 3328–50. https://doi.org/10.1002/anie.201409033

    Article  CAS  Google Scholar 

  10. Curran, K.A., Karim, A.S., Gupta, A., and Alper, H.S. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications, Metab. Eng., 2013, vol. 19, pp. 88–97. https://doi.org/10.1016/j.ymben.2013.07.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hernandez-Garcia, C.M. Finer, J.J., Identification and validation of promoters and cis-acting regulatory elements, Plant Sci., 2014, vol. 217, pp. 109–119.https://doi.org/10.1016/j.plantsci.2013.12.007

    Article  CAS  PubMed  Google Scholar 

  12. Li, T., Liu, B., Spalding, M.H., Weeks, D.P., and Yang, B., High-efficiency TALEN-based gene editing produces disease-resistant rice, Nat. Biotechnol., 2012, vol. 30, no. 5, p. 390–392. https://doi.org/10.1038/nbt.2199

    Article  CAS  PubMed  Google Scholar 

  13. Ndamukong, I., Abdallat, A.A., Thurow, C., Fode, B., Zander, M., Weigel, R., and Gatz, C., SA-inducible Arabidopsis glutaredoxin interacts with TGA factors and suppresses JA-responsive PDF1 2 transcription, Plant J., 2007, vol. 50, no. 1, pp. 128–139.https://doi.org/10.1111/j.1365-313X.2007.03039.x

    Article  CAS  PubMed  Google Scholar 

  14. Kay, R., Chan, A.M.Y., Daly, M., and McPherson, J., Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes, Science, 1987, vol. 236, no. 4806, pp. 1299–1302. https://doi.org/10.1126/sci-ence.236.4806.1299

    Article  CAS  PubMed  Google Scholar 

  15. Izumi, M., Tsunoda, H., Suzuki, Y., Makino, A., and Ishida., H., RBCS1A and RBCS3B, two major members within the Arabidopsis RBCS multigene family, function to yield sufficient Rubisco content for leaf photosynthetic capacity, J. Exp. Bot., 2012, vol. 63, pp. 2159–70. https://doi.org/10.1093/jxb/err434

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Blazeck, J., Alper, H.S., Promoter engineering: recent advances in controlling transcription at the most fundamental level, Biotechnol. J., 2013, vol. 8, no. 1, pp. 46–58. https://doi.org/10.1002/biot.201200120

    Article  CAS  PubMed  Google Scholar 

  17. Zhang, X.H., Webb, J., Huang, Y.H., Lin, L., Tang, R.S., and Liu, A., Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants, Plant Sci., 2011, vol. 180, no. 3, pp. 480–488. https://doi.org/10.1016/j.plantsci.2010.11.001

    Article  CAS  PubMed  Google Scholar 

  18. Umate, P., Genome-wide analysis of the family of light-harvesting chlorophyll a/b-binding proteins in Arabidopsis and rice, Plant Sign. Behav., 2010, vol. 5, no. 12, pp. 1537–1542.https://doi.org/10.4161/psb.5.12.13410

    Article  CAS  Google Scholar 

  19. Varchenko, O.I., Krasyuk, B.M., Fedchunov, O.O., Zimina, O.V., Parii M.F., and Symonenko, Yu.V., Genetic constructs creating using Golden Gate method, Fact. Exp. Evol. Organ., 2019, vol. 25, pp. 190–196.https://doi.org/10.7124/FEEO.v25.1163

    Article  Google Scholar 

  20. Bertani, G., Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli, J. Bacteriol., 1951, vol. 62, no. 3, pp. 293–300. PM-CID: PMC386127. PMID: 14888646. https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC386127/.

  21. Leuzinger, K., Dent, M., Hurtado, J., Stahnke, J., Lai, H., Zhou, X., and Chen, Q., Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins, JoVE, 2013, vol. 77, pp. 1–9. e50521. https://doi.org/10.3791/50521

  22. Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1989. https://archive.org/details/in.ernet.dli.2015.474251/ page/n53/mode/2up

  23. Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, vol. 72, pp. 248–254. https://doi.org/10.1006/abio.1976.9999

    Article  CAS  PubMed  Google Scholar 

  24. Ko, K. and Koprowski, H., Plant biopharming of monoclonal antibodies, Virus Res., 2005, vol. 111, no. 1, pp. 93–100. https://doi.org/10.1016/j.virusres.2005.03.016

    Article  CAS  PubMed  Google Scholar 

  25. Leuzinger, K., Dent, M., Hurtado, J., Stahnke, J., Lai, H., Zhou, X., and Chen, Q., Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins, JoVE, 2013, vol. 77, e50521. https://doi.org/10.3791/50521

    Article  CAS  Google Scholar 

  26. Shamloul, M., Trusa, J., Mett, V., and Yusibov, V., Optimization and utilization of Agrobacterium-mediated transient protein production in Nicotiana, JoVE, 2014, vol. 86, e51204. https://doi.org/10.3791/51204

    Article  CAS  Google Scholar 

  27. Conley, A.J., Zhu, H., Le, L.C., Jevnikar, A.M., Lee, B.H., Brandle, J.E., and Menassa, R., Recombinant protein production in a variety of Nicotiana hosts: a comparative analysis, Plant Biotechnol. J., 2011, vol. 9, no. 4, pp. 434–44. https://doi.org/10.1111/j.1467-7652.2010.00563.x

    Article  CAS  PubMed  Google Scholar 

  28. Wally, O., Jayaraj, J., and Punja, Z.K., Comparative expression of β-glucuronidase with five different promoters in transgenic carrot (Daucus carota L.) root and leaf tissues, Plant Cell Rep., 2008, vol. 27, no. 2, pp. 279–287. https://doi.org/10.1007/s00299-007-0461-1

    Article  CAS  PubMed  Google Scholar 

  29. Anuar, M.R., Ismail, I., and Zainal, Z., Expression analysis of the 35S CaMV promoter and its derivatives in transgenic hairy root cultures of cucumber (Cucumis sativus) generated by Agrobacterium rhizogenes infection, Afr. J. Biotechnol., 2011, vol. 10, no. 42, pp. 8236–8244. https://doi.org/10.5897/AJB11.130

    Article  CAS  Google Scholar 

  30. Patro, S., Kumar, D., Ranjan, R., Maiti, I.B., and Dey, N., The development of efficient plant promoters for transgene expression employing plant virus promoters, Mol. Plant, 2012, vol. 5, no. 4, pp. 941–944. https://doi.org/10.1093/mp/sss028

    Article  CAS  PubMed  Google Scholar 

  31. Li, Z., Jayasankar, S., and Gray, D.J., Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera), Plant Sci., 2001, vol. 160, no. 5, pp. 877–887. https://doi.org/10.1016/S0168-9452(01)00336-3

    Article  CAS  PubMed  Google Scholar 

  32. Elliot, A.R, Campbell, J.A, Dugdale, B., Brettell, R.I.S., and Grof, C.P.L., Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells, Plant Cell Rep., 1999, vol. 18, pp. 707–714. https://doi.org/10.1007/s002990050647

    Article  Google Scholar 

  33. Blumenthal, A., Kuznetzova, L., Edelbaum, O., Raskin, V., Levy, M., and Sela, I., Measurement of green fluorescent protein in plants: quantification, correlation to expression, rapid screening and differential gene expression, Plant Sci., 1999, vol. 142, no. 1, pp. 93–99. https://doi.org/10.1016/S0168-9452(98)00249-0

    Article  CAS  Google Scholar 

  34. Richards, H.A., Halfhill, M.D., Millwood, R.J., and Stewart, C.N.Jr., Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants, Plant Cell Rep., 2003, vol. 22, no. 2, pp. 117–121. https://doi.org/10.1007/s00299-003-0638-1

    Article  CAS  PubMed  Google Scholar 

  35. Zhou, X., Carranco, R, Vitha, S., and Hall, T.C., The dark side of green fluorescent protein, New Phytol., 2005, vol. 168, no. 2, pp. 313–322. https://doi.org/10.1111/j.1469-8137.2005.01489.x

    Article  CAS  PubMed  Google Scholar 

  36. Kapulnik, Y., Kahana, A., Bar-Akiva, A., Ben D.V.R., Wininger, S., and Ginzberg, I., US Patent no. 6844484, Washington, DC: U.S. Patent and Trademark Office, 2005.

  37. Cui, X.Y., Chen, Z.Y., Wu, L., Liu, X.Q., Dong, Y.Y., Wang, F.W. and Li, H.Y. RbcS SRS4 promoter from Glycine max and its expression activity in transgenic tobacco, Genet. Mol. Res., 2015, vol. 14, no. 3, pp. 7395–7405. https://doi.org/10.4238/2015

    Article  PubMed  Google Scholar 

  38. Tanabe, N., Tamoi, M., and Shigeoka, S., The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis, Gene, 2015, vol. 567, no. 2, pp. 244–250.https://doi.org/10.1016/j.gene.2015.05.006

    Article  CAS  PubMed  Google Scholar 

  39. Kushwah, N.S., Isolation, cloning and characterization of promoter of rubisco small subunit 2B (rbc-S2B) gene of Arabidopsis thaliana, Innovat. Farm., 2016, vol. 1, no. 4, pp. 119–28. http://www.innovativefarming.in/ index.php/innofarm/article/view/150.

  40. Dickinson, C.C., Weisberg, A.J., and Jelesko, J.G., Transient heterologous gene expression methods for poison ivy leaf and cotyledon tissues, Hort Sci., 2018, vol. 53, no. 2, pp. 242–246.https://doi.org/10.21273/HORTSCI12421-17

    Article  CAS  Google Scholar 

Download references

Funding

We would like to express our sincere gratitude to the National Academy of Sciences of Ukraine for providing financial support for this research in a departmental manner (State registration number U01174002589).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to O. I. Varchenko.

Ethics declarations

Conflict of interest. The authors declare that they have no conflict of interest, financial or otherwise.

Statement of compliance with standards of research involving humans as subjects. No animals or humans were used in the experiments in this study.

Additional information

Translated by K. Lazarev

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Varchenko, O.I., Kuchuk, M.V., Parii, M.F. et al. Comparison of gfp Gene Expression Levels after Agrobacterium-Mediated Transient Transformation of Nicotiana rustica L. by Constructs with Different Promoter Sequences. Cytol. Genet. 54, 531–538 (2020). https://doi.org/10.3103/S0095452720060110

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.3103/S0095452720060110

Keywords

Navigation