转基因作物中2种除草剂抗性基因aad1和dmo的PCR检测方法

Establishment of molecular detection of two herbicide-tolerant genes in genetically modified crops

【背景】抗除草剂转基因作物是全球种植面积最大的一类转基因植物,以除草剂抗性基因作为检测靶标的分子鉴定方法的研究与应用,对转基因生物安全的检测与监测有重要意义。【方法】根据除草剂抗性基因aad1和dmo的核苷酸序列设计PCR检测引物,并进行PCR反应体系优化、方法特异性、灵敏度、再现性等方面的测试,分别建立aad1基因和dmo基因的特异性PCR检测方法。【结果】建立的PCR检测方法在56~64℃的退火温度范围内均能获得一致性结果,具有良好的稳健性。该方法可将含有aad1基因和dmo基因的转基因作物与其他转基因作物区分开,其灵敏度可分别达到20个拷贝和40个拷贝。通过将aad1基因和dmo基因的检测引物放入同一管PCR反应体系中,还能在一次PCR中同时检测这2个靶标基因,双重PCR的检测灵敏度与单一PCR一致。【结论与意义】建立的分子方法可精准检测出含有aad1基因和dmo基因的转基因作物,具有特异性强、灵敏度高的特点,为抗除草剂转基因作物的筛选检测提供了可靠的技术支撑。

【Background】Herbicide-tolerant GM crops as a class of transgenic plants produce the global largest acreage of transgenic crops. For testing and monitoring of GMO safety,it is therefore important to develop methods for testing herbicide tolerant genes.【Method】Using the tolerent of herbicide resistance gene aad1 and dmo nucleotide,PCR primers were designed,and PCR reaction systems were optimized. The specificity,sensitivity,reproducibility tests of the methods were carried out. Hence the detection methods of aad1 and dmo gene-specific PCR were established respectively. 【Result】The PCR detection method was able to get stable and consistent results within the annealing temperature range of 56 ~ 64 ℃. The method could specifically identify crops containing aad1 or dmo gene among different kinds of transgenic crops with the detection sensitivity up to 20 copies or 40 copies. By mixing the detection primers of aad1 and dmo into the same PCR tube,a duplex PCR detection syatem was developed which would simultaneously amplify the two target genes in a single PCR system and with the same sensitivity as a single PCR. 【Conclusion and significance】The molecular methods developed in this study could accurately detect the genetically modified crops containing aad1 and dmo gene,with good specificity and high sensitivity. This method could provide a reliable technical support for screening and testing the herbicide-tolerant GM crops.