Mikrobiol. Z. 2020; 82(6):74-83.
doi: https://doi.org/10.15407/microbiolj82.06.074

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

O. Varchenko1,2, M. Kuchuk1, M. Parii2,3, Y. Symonenko1,2

1Institute of Cell Biology and Genetic Engineering, NAS of Ukraine
148 Acad. Zabolotny Str., Kyiv, 03143, Ukraine

2Ukrainian Scientific Institute of Plant Breeding
30 Vasylkivska Str., Kyiv, 03022, Ukraine

3National University of Life and Environmental Sciences of Ukraine
15 Heroyiv Oborony Str., Kyiv, 03041, Ukraine

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

Keywords: Nicotiana rustica L., molecular cloning, genetic constructs, terminators, green fluorescent protein, transient expression, spectrofluorometric analysis, protein analysis.

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  1. Lomonossoff G, D’Aoust M. Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Sci.2016; 353:1237–40. https://doi.org/10.1126/science.aaf6638
  2. Sharma A, Sharma M. Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv. 2009; 27:811–32. https://doi.org/10.1016/j.biotechadv.2009.06.004
  3. Nagaya S, Kawamura K, Shinmyo A, Kato K. The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant and cell physiology. 2010; 51(2):328–32. https://doi.org/10.1093/pcp/pcp188
  4. Moore M, Proudfoot N. Pre-mRNA processing reaches back to transcription and ahead to translation. Cell. 2009; 136(4):688–700. https://doi.org/10.1016/j.cell.2009.02.001
  5. Geisberg J, Moqtaderi Z, Fan X, Ozsolak F, Struhl K. Global analysis of mRNA isoform half-lives reveals stabilizing and destabilizing elements in yeast. Cell. 2014; 156:812−24. https://doi.org/10.1016/j.cell.2013.12.026
  6. Deaner M, Alper H. Promoter and terminator discovery and engineering. In: Synthetic Biology–Metabolic Engineering. Springer, Cham. Book; 2016. https://doi.org/10.1007/10_2016_8
  7. Giddings G, Allison G, Brooks D, Carter A. Transgenic plants as factories for biopharmaceuticals. Nat Biotechn. 2000; 18(11):1151–5. https://doi.org/10.1038/81132
  8. Du L, Gao R, Forster A. Engineering multigene expression in vitro and in vivo with small terminators for T7 RNA polymerase. Biotechnol Bioeng. 2009; 104:1189–96. https://doi.org/10.1002/bit.22491
  9. Du L, Villarreal S, Forster A. Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase. Biotechnol Bioeng. 2012; 109:1043–50. https://doi.org/10.1002/bit.24379
  10. Carter AD, Morris CE, McAllister W. Revised transcription map of the late region of bacteriophage T7 DNA. J Virol. 1981; 37:636–42. https://doi.org/10.1128/JVI.37.2.636-642.1981
  11. Curran K, Karim A, Gupta A, Alper H. 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; 19:88–97. https://doi.org/10.1016/j.ymben.2013.07.001
  12. Deaner M, Hal SA. Promoter and terminator discovery and engineering. Synt Biol–Metaboli Eng. Springer, Cham; 2016. p. 21–44. https://doi.org/10.1007/10_2016_8
  13. Chiasson D, Giménez-Oya V, Bircheneder M. A unified multi-kingdom Golden Gate cloning platform. Sci Rep. 2019; 9(1):1–12. https://doi.org/10.1038/s41598-019-46171-2
  14. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS. 2011; 6(2):e16765. https://doi.org/10.1371/journal.pone.0016765
  15. Engler C, Youles M, Gruetzner R, Ehnert T-M, Werner S, Jones, et al. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 2011; 3:839–43. https://doi.org/10.1021/sb4001504
  16. Werner S, Engler C, Weber E, Gruetzner R, Marillonnet S. Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system. Bioeng Bugs. 2012; 3(1):38–43. https://doi.org/10.4161/bbug.3.1.18223
  17. Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D. Green Fluorescent Protein as a Marker for Gene Expression. Science. 1994; 263:802–5. https://doi.org/10.1126/science.8303295
  18. Chiu W-L, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J. Engineered GFP as a vital reporter in plants. Cur Biol. 1996. 6:325–30. https://doi.org/10.1016/S0960-9822(02)00483-9
  19. Guilley H, Dudley R, Jonard G, Balàzs E, Richards K. Transcription of Cauliflower mosaic virus DNA: detection of promoter sequences and characterization of transcripts. Cell. 1982; 30:763–773. https://doi.org/10.1016/0092-8674(82)90281-1
  20. Dedonder A, Rethy R, Fredericq H, Van Montagu M, Krebbers E. Arabidopsis rbcS genes are differentially regulated by light. Plant Physiol. 1993; 101:801–8. https://doi.org/10.1104/pp.101.3.801
  21. Barker R, Idler K, Thompson D, Kemp J. Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol. 1983; 2:335–50. https://doi.org/10.1007/BF01578595
  22. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. Journal of visualized experiments: JoVE. 2007; 6:e253. https://doi.org/10.3791/253
  23. Lezin G, Kosaka Y, Yost HJ, Kuehn MR, Brunelli L. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles. PLoS One, 2011; 6:8. https://doi.org/10.1371/journal.pone.0023457
  24. Varchenko OI, Krasyuk BM, Fedchunov OO, Zimina OV, Parii MF, Symonenko YuV. Genetic constructs creating using Golden Gate method. Factors in experimental evolution of organisms. 2019; 25:190–6. https://doi.org/10.7124/FEEO.v25.1163
  25. Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd ed, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1989.
  26. Bertani G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951; 62(3):293–300. https://doi.org/10.1128/JB.62.3.293-300.1951
  27. Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q. Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins. 2013; 77:1–9. https://doi.org/10.3791/50521
  28. Bradford MM. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal Biochem. 1976; 72:248–54. https://doi.org/10.1016/0003-2697(76)90527-3
  29. Blakesly RW, Boezi JA. A new staining technique for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Anal Biochem. 1977; 82:580–2. https://doi.org/10.1016/0003-2697(77)90197-X
  30. Blumenthal A, Kuznetzova L, Edelbaum O, Raskin V, Levy M, Sela I. Measurement of green fluorescence protein in plants: quantification, correlation to expression, rapid screening and differential gene expression. Plant Science. 1999; 142:93–9. https://doi.org/10.1016/S0168-9452(98)00249-0
  31. Scholz O, Thiel A, Hillen W, Niederweis M. Quantitative analysis of gene expression with an improved green fluorescent protein. Europ J Biochem. 2000; 267(6):1565–70. https://doi.org/10.1046/j.1432-1327.2000.01170.x
  32. Naqvi RZ, Asif M, Saeed M, Asad S, Khatoon A, Amin I, Mukhtar Z, Bashir A, Mansoor S. Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants. Front Plant Sci. 2017; 8:55. https://doi.org/10.3389/fpls.2017.00055
  33. Hirt H, Kögl M, Murbacher T, Heberle-Bors E. Evolutionary conservation of transcriptional machinery between yeast and plants as shown by the efficient expression from the CaMV 35S promoter and 35S terminator. Current genetics. 1990; 17(6):473–9. https://doi.org/10.1007/BF00313074
  34. Eugster A, Murmann P, Kaenzig A, Breitenmoser A. Development and validation of a P-35S, T-nos, T-35S and P-FMV tetraplex real-time PCR screening method to detect regulatory genes of genetically modified organisms in food. CHIMIA Intern J Chem. 2014; 68(10):701–4. https://doi.org/10.2533/chimia.2014.701
  35. Schledzewski K, Mendel R. Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derived Emu and CaMV 35S in cells of barley, maize and tobacco. Transg Res. 1994; 3(4):249–55. https://doi.org/10.1007/BF02336778
  36. Outchkourov N, Peters J, De Jong J, Rademakers W, Jongsma M. The promoter–terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta. 2009; 216(6):1003–12. https://doi.org/10.1007/s00425-002-0953-8
  37. Ordon J, Bressan M, Kretschmer C, Dall’Osto L, Marillonnet S, Bassi R, Stuttmann J. Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation. Funct Integrat Genom. 2020; 20(1):151–62. https://doi.org/10.1007/s10142-019-00665-4