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.
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REFERENCES
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
Keasling, J.D., Manufacturing molecules through metabolic engineering, Science, 2010, vol. 330, no. 6009, pp. 1355–1358. https://doi.org/10.1126/science.1193990
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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/.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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.
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
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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).
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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
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DOI: https://doi.org/10.3103/S0095452720060110