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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 gmail.com

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


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