Selection of DNA Aptamers for Detection of CP4 EPSPS Proteinin GM Soybean

Abstract

Genetically modified (GM) soybean containing 5-enolpyruvylshikimate-3-phosphate synthase (epsps) gene from Agrobacterium tumefaciens strain CP4, which confers resistance to the herbicide glyphosate, have been found with the highest frequency in transgenic plants worldwide. Precise and rapid assay is required to detect the transgenic plants for monitoring the spread of transgenes in the environment. This study focused on the selection of DNA aptamers specific to the CP4 EPSPS protein which can be further applied in the development of GM test kit. The CP4 EPSPS protein (accession No. AAL67577.1) from GM soybean was
selected from Genbank and codon optimization was carried out for its gene expression in E. coli. Result showed that the recombinant CP4 EPSPS protein still positively reacted with the commercial antibody, therefore its native epitopes were conserved. This protein was used as an aptagen to select CP4 EPSPS specific DNA aptamers by systematic evolution of ligands by exponential enrichment (SELEX) method. Two DNA aptamers designated AptRR-A3 and AptRR-A5 were selected from the binding affinity test with GM soybean sap. The result showed the highest binding activity at an S/N ratio of 1.64 when using clone AptRR-A5 as capture aptamer and AptRR-A3 as detecting aptamer in sandwich ELAA. Sandwich ELAA was applied to detect CP4 EPSPS protein in three transgenic soybean seed samples and the results were in accordance with those from the commercial enzyme-linked immunosorbent assay (ELISA) test kit. However, the sensitivity of the developed sandwich ELAA in this research requires further improvement.