PECULIARITIES OF GREEN FLUORESCENT PROTEIN TRANSGENE DETECTION IN TOBACCO AND MAIZE PLANTS BY PCR І.О. Nitovska, D.Yu. Palekha, B.V. Morgun

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

cover biotech acta general

Biotechnologia Acta Т. 16, No. 4 , 2023
P. 43-48, Bibliography 16, Engl.
UDC: 602.6:577.213.3:577.112:582.926.2:633.15
DOI: https://doi.org/10.15407/biotech16.04.043

Full text: (PDF, in English)

PECULIARITIES OF GREEN FLUORESCENT PROTEIN TRANSGENE DETECTION IN TOBACCO AND MAIZE PLANTS BY PCR

І.О. Nitovska ¹, D.Yu. Palekha ², B.V. Morgun ¹

¹Institute of Cell Biology and Genetic Engineering, National Academy of Sciences of Ukraine, Kyiv
²ESC “Institute of Biology and Medicine” Taras Shevchenko National University of Kyiv

The work aimed to investigate the detection of different modifications of the green fluorescent protein gene (gfp) in the transgenic tobacco and maize plants by polymerase chain reaction (PCR).

Methods. Total DNA isolation, PCR, electrophoresis of DNA in agarose gel, bioinformatic resources.

Results. Three pairs of primers were used for PCR analysis of tobacco and maize containing wild-type gfp or mutant synthetic gene S65Tpgfp. The primer pair gfp1F-gfp1R interacted with the wild-type gfp gene only. The gfp2F-gfp2R primers interacted with the gfp gene of different modifications both in tobacco and maize. The gfp3F-gfp3R primer pair interacted with the modified S65Tpgfp gene in tobacco DNA but not with maize samples.

Conclusions. Primers for the detection of heterologous gfp gene, which were both narrowly specific (only one gene modification could be detected), and universal (more than one gene modification could be detected) were verified. It was shown that the primer pair gfp2F-gfp2R was universal for gfp genes detection both in tobacco and maize plants by PCR. The results obtained with gfp2F-gfp2R were reliably reproducible, so this primer pair is recommended for general use.

Key words: gfp, S65Tpgfp, Zea maize L., Nicotiana tabacum L., PCR, transgene detection, molecular markers.

References

ISAAA. Global status of commercialized biotech/GM crops in 2017: Biotech crop adoption surges as economic benefits accumulate in 22 years. Ithaca, NY: The International Service for the Acquisition of Agri-biotech Applications. [online], Available at: http://www.isaaa.org/resources/publications/briefs/53/download/isaaa-brief-53-2017.pdf
Grain: world markets and trade. Circular series FG-05-13[Internet]. Available at: http://www.fas.usda.com/psonline/circulars/grain.pdf.
Eurostat statistics explained. Agricultural production – crops. 2022, Available at: https://ec.europa.eu/eurostat/statistics-explained/index.php?title=Agricultural_production_-_crops
Lu A., Dichn S., Cigan M. Maize protein expression. Springer Science+Business Media, LLC 2015. Recent advancements in gene expression and enabling technologies in crop plants. 3–40. https://doi.org/10.1007/978-1-4939-2202-4_1
Chalfie M., Tu Y., Euskirchen G., Ward W.W., Prasher D.C. Green fluorescent protein as a marker for gene expression. 1994, 263, 802–805. https://10.1126/science.8303295
Varchenko O.I., Kuchuk M.V., Parii M.F., Symonenko Yu.V. Comparison of gfp gene expression levels after Agrobacterium-mediated transient transformation of Nicotiana rustica by constructs with different promoter sequences. Cytol Genet. 2020, 54, (6), 531–538. https://10.3103/S0095452720060110
Sindarovska Y., Kuchuk M. Long-term potato virus X (PVX)-based transient expression of recombinant GFP protein in Nicotiana benthamiana culture in vitro. Plants. 2021, 10, 2187–2207. https://3390/plants10102187
Pang S.-Z., DeBoer D.L., Wan Y., Ye G., Layton J.G., Neher M.K., Armstrong C.L., Fry J.E., Hinchee M.A., Fromm M.E. An improved green fluorescent protein gene as a vital marker in plants. Plant Physiol. 1996, 112(3), 893–900. https://1104/pp.112.3.893
Nitovska I.O., Vasylenko M.Yu., Morgun B.V. Effect of monocot introns on transgene expression in genus Nicotiana Biotechnol. Acta. 2018, 11(4), 69-79. https://doi.org/10.15407/biotech11.04.073
Stewart N.C. Jr., Via L.E. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR application. 1993, 14(5), 748–749.
QIAGEN CLC Main Workbench 6.9.2, Aarhus, Denmark.
Benson D.A., Cavanaugh M., Clark K., Karsch-Mizrachi I., Lipman D.J., Ostell J., Sayers E.W. Nucleic Acids Res. 2013, 41(Database issue), D36-42. https://doi.org/10.1093/nar/gks1195
Lambert T.J. FPbase: a community-editable fluorescent protein database. Nature Methods. 2019, 16, 277–278. Available at: https://www.fpbase.org/protein/egfp/
Lipp Joаo K.H., Brown T.A. Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58C1Rif(r)::pGSFR1161 in the presence of acetosyringone. Plant Cell Rep. 1993, 12, 422–425.https://doi.org/10.1007/BF00234705
Brody J.R., Kern S.E. History and principles of conductive media for standard DNA electrophoresis. Biochem. 2004, 333, 1–13. https://10.1016/j.ab.2004.05.054
Kalle E., Kubista M., Rensing Ch. Multi-template polymerase chain reaction. Detect. Quantif. 2014, 2, 11‒29. https://10.1016/j.bdq.2014.11.002