抗草甘膦转基因大豆PCR检测方法的建立与应用-农药学专业毕业论文.docxVIP

堡圭耋堡堡塞 堡圭耋堡堡塞 垫鐾 壅錾壅些套耋 摘 要 转基因作物及食品的检测是食品标识和法律实施所必需的。本研究以抗草甘膦转基 因大豆为实验材料，进行检测方法研究。目的是为制定转基因大豆及豆粕的检测规程提 供依据，为我国转基因产品管理制度的建立与实施提供可行的检测方法。 主要的研究结果如下： 1．建立了一套大豆种子、叶片检测抗草甘膦转外源基因caMv35s启动子、NOS终止 子和目的基因CP4一EPSPS的PCR检测方法，并通过Southern杂交进一步证实了PCR检 测结果的可靠性。 ／ 2．根据大豆凝集素基因设计引物，做内置检测标准。慨功的优化了一套快速、准 确、灵敏、可靠的抗草甘膦转基因大豆PCR检测方法。适用于对进口的大豆及大豆粗加 1=产品豆粕的检测。r一， ，， 3．确定双重水和非转基因大豆种子混合稀释最低检测限分别为0．01％和0 05％。 4．利用建立的检测方法，对转基因情况未知的进口大豆种子和豆粕共6个样品进 行检测．滇中5个样品检测出具有CaMV35S启动子、NOS终止子、CP4一EPSPS基因成分， 而在国内非转基因大豆中未检测出上述成分。卜r～ 5．将建立的检测方法应用于抗草甘膦转基因大豆京引D一1×非转基因大豆豫豆12 杂交R群体(共30株)检测，陕田施药对草甘膦为抗性的植株，PCR检测为阳性，而敏 感株，PCR检测为阴性，符合率为100％。广F一 6．应用优化的检测方法对京引D一1×豫豆12 Fs代群体进行检测。鲐果表明，CaMV35S 启动子、NOS终止子和CP4一EPSPS基因三种元件的PCR检测结果一致，PCR检测和大田旌 药检测符合率为93．O％。这进一步说明了检测方法的可靠性。表型鉴定结果表明，抗性 基因与种脐颜色无连锁关系。户厂一 关键词： 草甘膦 转基因大豆PCR检测 AbstractThe Abstract The detection of genetically modified crops(GMC)is becoming both food labels and legal necessity．The purpose of this study is to establish GMC detection method using PCR technology Firstly,primers were designed to amplify parts of the 35S promoter,the NOS terminator,CP4一EPSPS target DNA and lectin，lectin primer is eo-amplified together with the target DNA specially used as internal standards in the same PCR tube．Secondly,PCR result is to be conformed by Southern hybridization．Thirdly,genetically modified soybean DNA is mixed with ddH20 with different ratios or genetically modified soybean(GMS)seeds is mixed with non—genetically modified soybean(non-GMS)seeds，which indicate that the PCR detection methods established sensitive． Fourthly,PCR method is used to detect the generation of the hybridization population(F2 and F2：s)of GMS and non-GMS as well as imported soybean seeds and bean meal coming from different countries，which show whether spraying glyphosate is consistent with PCR detection or not,whether resistant to giyphosate gene is linked with hilum color or not． The results obtained aS followings： PCR method was established to detect GMS seed and leaf resistant to glyphosate and Sou