Test system for the identification of soybean line GTS 40-3-2

Abstract

Background. Currently about 34 transgenic soybean lines are registered in the world, among them 26 are resistant to herbicides, 5 to insecticides, 8 are modified to have new qualities, concerning fatty acid composition, resistance to antibiotics, presence of visual marker and other. All these transgenically modified lines of soybean are used as foodstuffs or food supplements.
As product properties are caused by specific genetic insertions, determination of GMOs should include not only identification of their overall regulatory elements, but at the same time of specific genetic constructions.
The aim of the study is to develop specific primers for the simultaneous identification of regulatory elements and transformational events of soybean GTS 40-3-2.
Material and methods. Objects of the study were as follows: DNA of genetically modified and unmodified soya, DNA of genetically modified and unmodified tobacco, DNA of plasmid pUC57, which contains modified gene EPSPS of Agrobacterium tumefaciens with transformation event GTS 40-3-2. DNA isolation from soybeans and tobacco were performed by "Ukrmetrteststandart" systems. Primers developed by the author were used for polymerase chain reaction
Results. Based on the analyzed information on the detection and identification of the transgene DNA sequence in plants material, in soya in particular, we have developed primers of own design for simultaneous identification of GM soybean inserts: 35S, NOS, species-specific lectin gene, gene sequence of transformational event Roundup Ready Soybean GTS 40-3-2 and CP4 EPSPS gene, which contains a complete copy of the enol pyruvilshykimat phosphate synthetase gene from soil bacterium Agrobacterium sp. strain CP4, transferred into the soy genome encoding the enzyme EPSPS, which determines resistance to the herbicide glyphosate. Pairs of primers obtained are flanking DNA fragments of the following sizes: 103 bp for lectin gene; 183 bp for CP4 EPSPS gene; 126 bp for the event Roundup Ready Soybean GTS 40-3-2; 263 bp for the 35S promoter and 126 bp for NOS-terminator. To identify the CP4 EPSPS gene in genetically modified and unmodified soy, in genetically modified and unmodified tobacco, and in plasmid pUC57, which contains the modified EPSPS gene of Agrobacterium tumefaciens, PCR analysis has been carried out with subsequent separation of the amplicon obtained by electrophoresis on 2 % agarose gel. The expected lengths of amplification products obtained at 60 °C, correspond to the primers applied: lectin gene – 103 bp, CP4 EPSPS – 183 bp, 35S promoter – 263 bp and NOS-terminator – 169 bp.
Conclusion. The developed primers for the gene lectin, the gene CP4 EPSPS, the specific event RRS GTS 40-3-2, for the promoter 35S and NOS-terminator could be used for the simultaneous detection of genetic modifications of soybean and the modifications in other crops if they contain aforementioned structures. It is shown that identifying of the transformational event GTS 40-3-2 in food does not always coincide with the presence of 35S promoter. The presence of NOS-terminator in samples that showed a lack of event GTS 40-3-2, may indicate the presence of other GM designs or other transformational event soybeans in their composition.