College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan Province, 410128, China; Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province, 310021, China.
Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province, 310021, China; Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang Province, 310021, China.
Anal Biochem. 2025 Jan;696:115669. doi: 10.1016/j.ab.2024.115669. Epub 2024 Sep 10.
Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16-256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %-7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44-445.80 μg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.
草甘膦抗性是转基因（GM）作物的一个非常重要的特性。水稻 EPSPS 基因的突变导致高水平的草甘膦抗性，为开发草甘膦耐受作物提供了巨大的潜力。水稻对草甘膦的抗性与抗性基因的表达水平有关。因此，开发一种方便、稳定、敏感的定量检测 OsmEPSPS 蛋白的方法对于开发草甘膦抗性作物至关重要。我们使用特异性单克隆抗体（mAb）捕获 OsmEPSPS 和 HRP 标记的抗 OsmEPSPS 兔多克隆抗体（pAb），开发了双抗体夹心定量 ELISA（DAS-ELISA）。该方法可用于检测 16-256ng/mL 线性范围内的 OsmEPSPS，具有良好的批内和批间重复性（%CV 值：0.17%-7.24%）。使用 DAS-ELISA 定量检测了转基因水稻品系（54.44-445.80μg/g）中的 OsmEPSPS 表达。此外，还通过 Western blot 验证了 OsmEPSPS 基因的表达。这项研究证明了 DAS-ELISA 用于检测 GM 水稻中 OsmEPSPS 的可靠性和稳定性。
