Comparison of gfp Gene Expression Levels after Agrobacterium-Mediated Transient Transformation of Nicotiana rustica L. by Constructs with Different Promoter Sequences

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.