OsDREB3转基因大豆品系特异性检测方法的研究
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摘要
转基因食品的安全性及对生态环境的影响等问题一直是关注的焦点。目前商业化的转基因大豆主要为抗草甘膦大豆,抗逆基因的转基因大豆因地理环境及全球商业化的需求的增多,商业化的呼声也越来越高,但迄今为止我国尚未建立抗逆转基因大豆的检测技术。
本实验以已进入环境释放或生产性试验、或获得安全证书的转基因植物环境安全评价阶段的耐低温抗逆转基因大豆新品系转基因大豆OsDREB3为研究对象,利用染色体步移技术,获得了转基因大豆OsDREB35'端和3’端旁侧序列信息。通过生物信息学分析方法明确了5’端旁侧序列344bp覆盖了转化载体及转基因大豆基因组,在大豆基因组为1号染色体,位置为49,828,108～49,836,357；3’端旁侧序列共长2189bp,属未知基因序列。
本实验根据已得到的5’端旁侧序列信息,建立了转基因大豆OsDREB3品系特异性定性和定量检测方法。品系特异性检测定性和定量引物所扩增片段覆盖了大豆基因组及转化载体序列信息,在检测过程中随机选择了4种转基因大豆,2种转基因玉米,2种转基因水稻同时进行品系特异性定性及定量检测,结果显示除转基因大豆OsDREB3有扩增片段或扩增曲线外,其余转基因作物均没有扩增片段或扩增曲线,表明该定性及定量检测方法符合品系特异性检测要求。定性检测扩增片段大小为160bp。检测灵敏度为0.1%,比欧盟检测下限(0.9%)低,且重现性好,可用于转基因大豆OsDREB3定性检测。所设计的荧光定量PCR引物,扩增曲线检测下限可达0.01%。
建立了转基因大豆多重PCR检测方法和转基因大豆OsDREB3巢式PCR检测方法。本实验选择了转基因大豆GTS40-3-2、转基因大豆A2704-12及转基因大豆OsDREB3DNA作为待检测基因模板,对多重PCR体系中的成分进行了多次调整,尝试了多种不同引物对的配比,扩增片段大小相差100多个碱基,范围从700bp～200bp之间,可视性好。最终确定了最佳检测方案,成功实现了3种转基因大豆、4组不同片段的同时扩增和检测,检测下限可达0.01%。巢式PCR所用引物在满足品系特异定性检测的同时,也要提高其检测的灵敏度,本实验所建立巢式PCR检测方法,检测灵敏度可达0.005%。
本实验在原有构建有4种转基因大豆品系特异性序列的标准分子的基因上又构建了转基因大豆OsDREB3品系特异性序列,新构建的含有5种转基因大豆品系特异性序列及大豆内参基因lectin的阳性对照标准分子,本实验通过实时荧光定量PCR方法,以大豆凝集素基因(lectin)作为内源参照基因,确定了外源基因OsDREB3在转基因大豆OsDREB3基因组为单拷贝作物。
本实验希望通过染色体步移技术得到的OsDREB3外源基因旁翼序列,建立起来的转基因大豆OsDREB3一整套灵敏高、重现性好、可信度高、特异性强的品系特异性检测体系,充分提高了转基因大豆检测工作效率,从而完善了现有转基因大豆标准分子在OsDREB3抗旱基因检测方面的不足,为我国建立转基因大豆OsDREB3标识管理制度提供参考。该方法为建立转基因大豆OsDREB3标准体系提供技术支持,并为转基因产品标识制度的规范提供有价值的参考。
Safety and impaci on the ecological environment of genetically modified foods has been the focus of attention. Most of the commercialized of genetically modified soybeans is glyphosate-resistant soybean, stress resistance genes due to the geographical environment and the global commercialization of the increase in demand for the commercialization are getting higher and higher, but so far there has not been established the detection techniques resilience gene of genetically modified soybeans in China.
In this study, we set up the detecting techniques for specificity of transgenic soybean OsDREB3due to the51end and3'end flanking sequence information of genetically modified soybean OsDREB3, which had entered the environmental releasing or production test or got the security certification of environmental safety evaluation for genetically modified plants. The5'end flanking sequence of344bp covering the transformation vectors and transgenic soybean gene group was gained by chromosome walking technique, in the soybean genome on chromosome1, position for49,828,108-49,836,357;3' end flanking sequence of a total length2189bp, the case of unknown gene sequences.
The experiment was based on the5' end flanking sequence information, specific qualitative and quantitative detection of genetically modified soybeans OsDREB3strains, event-specific detection of qualitative and quantitative cited material by amplified fragment covering soybean genome and the transformation of vector sequence information, in the detection process randomly select the four kinds of genetically modified soybean, were randomly selected two kinds of genetically modified com, two kinds of transgenic rice at the same time, event-specific qualitative and quantitative detection were carried out results showing that in addition to transgenic soybeans OsDREB3in the amplified fragment or outside of the amplification curve, the rest of genetically modified crops have not amplified fragments or amplification curve, indicating that the qualitative and quantitative detection methods to comply with the requirements of the event-specific detection. Qualitative detection of amplified fragment size of160bp. The detection sensitivity of0.1%than the EU detection limit (0.9%) and good reproducibility can be used to genetically modified soybeans OsDREB3qualitative detection. The design of quantitative PCR primers, amplification curve detection limit up to0.01%.
multiplex PCR assay for genetically modified soybeans and transgenic soybean OsDREB3nested PCR method were set up. In this study, to select the transfected gene soybean GTS40-3-2, genetically modified soybean A2704-12and transgenic soybean OsDREB3DNA as to be detected gene template in multiplex PCR components were adjusted several times, a variety of different primers were tried on the ratio The amplified fragment size difference of more than100bases, ranging from700bp to200bp between good visibility. The best detection scheme is finalized, the successful implementation of three kinds of genetically modified soybeans, amplification and detection of four different fragments, The low limits of detection was0.01%. Nested PCR primers to meet the event-specific qualitative detection, but also to improve the detection sensitivity, this experiment created by the nested PCR method, and the sensitivity of the detection was0.005%.
In this study, we constructed a standard molecular containing five kinds of transgenic soybean lines and soybean internal reference gene lection as the positive standard control. In this study, by real-time quantitative PCR, the soybean lectin gene (the lectin) as the endogenous reference gene to determine the crop exogenous gene OsDREB3in the genetically modified soybean OsDREB3genome. We found the OsDDREB3in soybean OsDreb3was only one copy.
In this experiment, we built up a set of sensitive and high, with good reproducibility, high reliability, specificity and event-specific detection system of genetically modified soybean OsDREB3, by which we can improve detection efficiency and perfect insufficient of detection in modified soybean OsDREB3, and provide technical support for the detection of the the transgenic soybean OsDREB3, the results of this study provide a valuable reference for GMO labeling system specification.
本实验以已进入环境释放或生产性试验、或获得安全证书的转基因植物环境安全评价阶段的耐低温抗逆转基因大豆新品系转基因大豆OsDREB3为研究对象,利用染色体步移技术,获得了转基因大豆OsDREB35'端和3’端旁侧序列信息。通过生物信息学分析方法明确了5’端旁侧序列344bp覆盖了转化载体及转基因大豆基因组,在大豆基因组为1号染色体,位置为49,828,108～49,836,357；3’端旁侧序列共长2189bp,属未知基因序列。
本实验根据已得到的5’端旁侧序列信息,建立了转基因大豆OsDREB3品系特异性定性和定量检测方法。品系特异性检测定性和定量引物所扩增片段覆盖了大豆基因组及转化载体序列信息,在检测过程中随机选择了4种转基因大豆,2种转基因玉米,2种转基因水稻同时进行品系特异性定性及定量检测,结果显示除转基因大豆OsDREB3有扩增片段或扩增曲线外,其余转基因作物均没有扩增片段或扩增曲线,表明该定性及定量检测方法符合品系特异性检测要求。定性检测扩增片段大小为160bp。检测灵敏度为0.1%,比欧盟检测下限(0.9%)低,且重现性好,可用于转基因大豆OsDREB3定性检测。所设计的荧光定量PCR引物,扩增曲线检测下限可达0.01%。
建立了转基因大豆多重PCR检测方法和转基因大豆OsDREB3巢式PCR检测方法。本实验选择了转基因大豆GTS40-3-2、转基因大豆A2704-12及转基因大豆OsDREB3DNA作为待检测基因模板,对多重PCR体系中的成分进行了多次调整,尝试了多种不同引物对的配比,扩增片段大小相差100多个碱基,范围从700bp～200bp之间,可视性好。最终确定了最佳检测方案,成功实现了3种转基因大豆、4组不同片段的同时扩增和检测,检测下限可达0.01%。巢式PCR所用引物在满足品系特异定性检测的同时,也要提高其检测的灵敏度,本实验所建立巢式PCR检测方法,检测灵敏度可达0.005%。
本实验在原有构建有4种转基因大豆品系特异性序列的标准分子的基因上又构建了转基因大豆OsDREB3品系特异性序列,新构建的含有5种转基因大豆品系特异性序列及大豆内参基因lectin的阳性对照标准分子,本实验通过实时荧光定量PCR方法,以大豆凝集素基因(lectin)作为内源参照基因,确定了外源基因OsDREB3在转基因大豆OsDREB3基因组为单拷贝作物。
本实验希望通过染色体步移技术得到的OsDREB3外源基因旁翼序列,建立起来的转基因大豆OsDREB3一整套灵敏高、重现性好、可信度高、特异性强的品系特异性检测体系,充分提高了转基因大豆检测工作效率,从而完善了现有转基因大豆标准分子在OsDREB3抗旱基因检测方面的不足,为我国建立转基因大豆OsDREB3标识管理制度提供参考。该方法为建立转基因大豆OsDREB3标准体系提供技术支持,并为转基因产品标识制度的规范提供有价值的参考。
Safety and impaci on the ecological environment of genetically modified foods has been the focus of attention. Most of the commercialized of genetically modified soybeans is glyphosate-resistant soybean, stress resistance genes due to the geographical environment and the global commercialization of the increase in demand for the commercialization are getting higher and higher, but so far there has not been established the detection techniques resilience gene of genetically modified soybeans in China.
In this study, we set up the detecting techniques for specificity of transgenic soybean OsDREB3due to the51end and3'end flanking sequence information of genetically modified soybean OsDREB3, which had entered the environmental releasing or production test or got the security certification of environmental safety evaluation for genetically modified plants. The5'end flanking sequence of344bp covering the transformation vectors and transgenic soybean gene group was gained by chromosome walking technique, in the soybean genome on chromosome1, position for49,828,108-49,836,357;3' end flanking sequence of a total length2189bp, the case of unknown gene sequences.
The experiment was based on the5' end flanking sequence information, specific qualitative and quantitative detection of genetically modified soybeans OsDREB3strains, event-specific detection of qualitative and quantitative cited material by amplified fragment covering soybean genome and the transformation of vector sequence information, in the detection process randomly select the four kinds of genetically modified soybean, were randomly selected two kinds of genetically modified com, two kinds of transgenic rice at the same time, event-specific qualitative and quantitative detection were carried out results showing that in addition to transgenic soybeans OsDREB3in the amplified fragment or outside of the amplification curve, the rest of genetically modified crops have not amplified fragments or amplification curve, indicating that the qualitative and quantitative detection methods to comply with the requirements of the event-specific detection. Qualitative detection of amplified fragment size of160bp. The detection sensitivity of0.1%than the EU detection limit (0.9%) and good reproducibility can be used to genetically modified soybeans OsDREB3qualitative detection. The design of quantitative PCR primers, amplification curve detection limit up to0.01%.
multiplex PCR assay for genetically modified soybeans and transgenic soybean OsDREB3nested PCR method were set up. In this study, to select the transfected gene soybean GTS40-3-2, genetically modified soybean A2704-12and transgenic soybean OsDREB3DNA as to be detected gene template in multiplex PCR components were adjusted several times, a variety of different primers were tried on the ratio The amplified fragment size difference of more than100bases, ranging from700bp to200bp between good visibility. The best detection scheme is finalized, the successful implementation of three kinds of genetically modified soybeans, amplification and detection of four different fragments, The low limits of detection was0.01%. Nested PCR primers to meet the event-specific qualitative detection, but also to improve the detection sensitivity, this experiment created by the nested PCR method, and the sensitivity of the detection was0.005%.
In this study, we constructed a standard molecular containing five kinds of transgenic soybean lines and soybean internal reference gene lection as the positive standard control. In this study, by real-time quantitative PCR, the soybean lectin gene (the lectin) as the endogenous reference gene to determine the crop exogenous gene OsDREB3in the genetically modified soybean OsDREB3genome. We found the OsDDREB3in soybean OsDreb3was only one copy.
In this experiment, we built up a set of sensitive and high, with good reproducibility, high reliability, specificity and event-specific detection system of genetically modified soybean OsDREB3, by which we can improve detection efficiency and perfect insufficient of detection in modified soybean OsDREB3, and provide technical support for the detection of the the transgenic soybean OsDREB3, the results of this study provide a valuable reference for GMO labeling system specification.
引文
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