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Comparison of the GFP gene expression levels after Agrobacterium-mediated transient transformation of Nicotiana rustica L. by constructions with different promotor sequences

Varchenko O.I., Kuchuk M.V., Parii M.F., Symonenko Yu.V.


SUMMARY. Promoters are key elements on the level of gene expression, so their selection is an important step in genetic engineering research. The reporter gfp gene, which encodes green fluorescent protein (GFP), was transiently expressed in the leaf tissues of the Aztec tobacco Nicotiana rustica L. Compared to other species of the Nicotiana genus, it has a large potential for the 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 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 7th day after infiltration. The highest level of expression was obtained using the double 35S promoter (D35S CaMV) and the lowest when the promoter of the LHB1B1 gene was used.

Key words: Aztec tobacco, Nicotiana rustica L., promoter, gfp gene, green fluorescent protein (GFP), transient expression, genetic constructs, spectrofluorimetry analysis, quantitative protein analysis

Tsitologiya i Genetika 2020, vol. 54, no. 6, pp. 35-44

  • Institute of Cell Biology and Genetic Engineering, NAS of Ukraine, Kyiv
  • All-Ukrainian Scientific Institute of Breeding, Kyiv
  • National University of Life and Environmental Sciences of Ukraine, Kyiv

E-mail: okvarchenko

Varchenko O.I., Kuchuk M.V., Parii M.F., Symonenko Yu.V. Comparison of the GFP gene expression levels after Agrobacterium-mediated transient transformation of Nicotiana rustica L. by constructions with different promotor sequences, Tsitol Genet., 2020, vol. 54, no. 6, pp. 35-44.

In "Cytology and Genetics":
O. I. Varchenko, M. V. Kuchuk, M. F. Parii & Yu. V. Symonenko Comparison of gfp Gene Expression Levels after Agrobacterium-Mediated Transient Transformation of Nicotiana rustica L. by Constructs with Different Promoter Sequences, Cytol Genet., 2020, vol. 54, no. 6, pp. 531538
DOI: 10.3103/S0095452720060110


1. Blazeck, J. and Alper, H., Systems metabolic engineering: genome-scale models and beyond, Biotechnol. J., 2010, vol. 5, no. 7, pp. 647659.

2. Keasling, J.D., Manufacturing molecules through metabolic engineering, Science, 2010, vol. 330, no. 6009, pp. 13551358.

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. 117.

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. 111. doi. org/

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. 113128.

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. 460471.

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. 182188.

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. 158174.

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. 332850.

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. 8897.

11. Hernandez-Garcia, C.M. Finer, J.J., Identification and validation of promoters and cis-acting regulatory elements, Plant Sci., 2014, vol. 217, pp. 109119.

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. 390392.

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. 128139.

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. 12991302.

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. 215970.

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. 4658.

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. 480488.

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. 15371542.

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. 190196.

20. Bertani, G., Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli, J. Bacteriol., 1951, vol. 62, no. 3, pp. 293300. PM-CID: PMC386127. PMID: 14888646. 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. 19. e50521.

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. 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. 248254.

24. Ko, K. and Koprowski, H., Plant biopharming of monoclonal antibodies, Virus Res., 2005, vol. 111, no. 1, pp. 93100.

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.

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.

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. 43444.

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. 279287.

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. 82368244.

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. 941944.

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. 877887.

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. 707714.

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. 9399.

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. 117121.

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. 313322.

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. 73957405.

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. 244250.

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. 11928. 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. 242246.

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