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Selection of DNA Aptamers for Detection of CP4 EPSPS Proteinin GM Soybean |
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| รหัสดีโอไอ | |
| Creator | 1. Adcharapun Chaicharoen 2. Ratchanee Hongprayoon |
| Title | Selection of DNA Aptamers for Detection of CP4 EPSPS Proteinin GM Soybean |
| Publisher | Department of Agriculture |
| Publication Year | 2561 |
| Journal Title | Thai Agricultural Research Journal |
| Journal Vol. | 36 |
| Journal No. | 2 |
| Page no. | 141-152 |
| Keyword | Glyphosate, Enzyme-linked oligonucleotide assay, ELONA, Biotech soybean, ไกลโฟเสท, เอนไซม์ลิงค์โอลิโกนิวคลีโอไทด์แอสเซย์, อีโลน่า, ถั่วเหลืองไบโอเทค, ????????????????? |
| URL Website | http://at.doa.go.th/journal |
| Website title | Thai Agricultural Research Journal |
| ISSN | 0125-8389 |
| Abstract | Genetically modified (GM) soybean containing 5-enolpyruvylshikimate-3-phosphatesynthase (epsps) gene from Agrobacterium tumefaciens strain CP4, which confers resistanceto the herbicide glyphosate, have been found with the highest frequency in transgenic plantsworldwide. Precise and rapid assay is required to detect the transgenic plants for monitoringthe spread of transgenes in the environment. This study focused on the selection of DNAaptamers specific to the CP4 EPSPS protein which can be further applied in the developmentof GM test kit. The CP4 EPSPS protein (accession No. AAL67577.1) from GM soybean wasselected from Genbank and codon optimization was carried out for its gene expression inE. coli. Result showed that the recombinant CP4 EPSPS protein still positively reacted withthe commercial antibody, therefore its native epitopes were conserved. This protein wasused as an aptagen to select CP4 EPSPS specific DNA aptamers by systematic evolution ofligands by exponential enrichment (SELEX) method. Two DNA aptamers designated AptRR-A3and AptRR-A5 were selected from the binding affinity test with GM soybean sap. The resultshowed the highest binding activity at an S/N ratio of 1.64 when using clone AptRR-A5 ascapture aptamer and AptRR-A3 as detecting aptamer in sandwich ELAA. Sandwich ELAAwas applied to detect CP4 EPSPS protein in three transgenic soybean seed samplesand the results were in accordance with those from the commercial enzyme-linkedimmunosorbent assay (ELISA) test kit. However, the sensitivity of the developed sandwichELAA in this research requires further improvement. |
