Production of marker-free tomato plants expressing the supersweet protein thaumatin II gene under the control of predominantly fruit-specific promoters
Despite the lack of evidence of the danger of genetically modified organisms the presence of marker and antibiotic-resistant genes in transgenic plants causes concern to consumers. Genetically modified plants with viral and bacterial genes are adopted by consumers, but with concerns; in addition, constitutive promoters have a number of disadvantages in industrial-scale cultivation of plants. In our study, we used the pMF vector system (Wageningen Plant Research, Wageningen, Netherlands), which combines inducible site-specific recombinase and a bifunctional selectable gene to obtain marker-free tomato plants. The gene of interest was the supersweet thaumatin II protein from the tropical plant Thaumatococcus daniellii under the control of tomato predominantly fruit-specific early-light inducible protein (ELIP) or E8 promoters and tomato Rubisco terminator. The use of this gene in our laboratory allowed enhancing sweetness, as well as improving the taste characteristics of fruit such as apple, strawberries, carrots, tomatoes, and pears. By using different strategies of early and delayed selection we developed a protocol for obtaining fully marker-free tomato plants, which was checked by polymerase chain reaction and Southern blotting. The thaumatin II gene expression was confirmed by reverse transcription-PCR and western blotting analyses. The fruit of transgenic and marker-free tomato plants displayed a sweet taste. A quantitative comparative assessment of the level of expression of the thaumatin protein under the control of two promoters was carried out using enzyme-linked immunosorbent assay. Multiple and/or incomplete T-DNA inserts that often occur during transformation of Solanaceae greatly reduced the efficiency of the system used.
The strong tomato ELIP promoter provides a high level of expression of the supersweet thaumatin II protein gene in the fruit of marker-free tomato plants.
KeywordsSolanum lycopersicum E8 Early-light inducible protein (ELIP) Cytosine deaminase R/RS recombination system High expression level
One-way analysis of variance
Cauliflower mosaic virus 35S promoter
Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9
Early-light inducible protein
Enzyme-linked immunosorbent assay
Heat shock protein
Murashige and Skoog culture medium
Phosphate buffer saline
Sodium dodecyl sulfate
Transcription activator-like effector nucleases
Total soluble protein
We thank O. Kozlov, L. Shaloiko and A. Firsov for the western blot analysis. This research was carried out using the unique scientific installation “Fitotron” (registration no. 2-2.9).
VT designed the study, constructed the transformation vectors, obtained transgenic and marker-free tomato plants, performed the PCR and RT-PCR, and wrote the manuscript. AP performed the Southern blot and ELISA. SD supervised the project and provided financial support. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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