转基因耐除草剂大豆ZH10-6多重PCR检测方法的建立  

Establishment of Multiplex PCR Detection Method for Genetically Modified Herbicide Tolerant Soybean(Glycine max)ZH10-6

作　　者：李瑞环 刘双 兰青阔[1] 曹际娟 王永[1] 赵新[1] LI Rui-Huan;LIU Shuang;LAN Qing-Kuo;CAO Ji-Juan;WANG Yong;ZHAO Xin(Biotechnology Research Institute,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China;College of Life Sciences,Dalian Minzu University,Dalian 116600,China)

机构地区：[1]天津市农业科学院生物技术研究所,天津300381 [2]大连民族大学生命科学学院,大连116600

出　　处：《农业生物技术学报》2021年第8期1640-1648,共9页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育专项(2019ZX08013002-004;2019ZX08004001-005)。

摘　　要：转基因耐除草剂大豆(Glycine max)ZH10-6是由中国农业科学院作物科学研究所研究的转草甘膦降解基因(glyphosate acetyltransferase,GAT)和草甘膦抗性基因(pseudomonas fluorescens G2 strain 5-enolpyruvyl shikimate-3-phosphate synthase,G2-EPSPS)的耐除草剂转基因大豆新品系。为建立其分子特征转化体特异性的检测方法,本研究以插入基因GAT、G2-EPSPS和大豆基因组的边界位点为靶标,设计4组插入序列边界位点特异性引物,结合大豆内标准基因Lectin进行多重PCR(multiplex PCR,MPCR)研究。通过引物特异性测试、体系和反应程序优化、灵敏度分析、以及世代遗传稳定性应用,建立了转基因大豆ZH10-6分子特征转化体特异性多重PCR检测方法。结果表明,该方法具有良好的特异性,MPCR检测体系中5种靶标(边界A,边界B,边界C,边界D,内源基因Lectin)的最适引物终浓度(μmol/L)配比为:0.8∶0.3∶0.1∶0.2∶0.05。每种靶标的检测灵敏度可达到1%,可在3个世代间稳定遗传,适用于ZH10-6插入位点快速高效检测。本研究建立的检测方法可有效提高检测效率,降低检测成本,为转基因耐除草剂大豆ZH10-6新品种的分子特征转化体特异性鉴定提供新的检测技术,为该品种分子特征转化体的生物安全评价及知识产权保护和市场行政监管提供技术支撑。Herbicide-tolerant transgenic soybean(Glycine max)ZH10-6 is a new herbicide-tolerant genetically modified soybean line with glyphosate degradation gene(glyphosate acetyltransferase,GAT)and glyphosate resistance gene(pseudomonas fluorescens G2 strain 5-enolpyruvyl shikimate-3-phosphate synthase,G2-EPSPS)studied by the Institute of Crop Science,Chinese Academy of Agricultural Sciences.In order to establish anevent-specific detection method with it,multiplex PCR(MPCR)reaction with 4 sets of PCR primers covering different regions of the insertion and border sequence and 1 primer set for the soybean internal standard gene Lectin were evaluated..Through primer-specific testing,optimization of PCR reaction system and procedure,assessment of reaction sensitivity and stability for the different generations of the transgenic soybean,a multiplex PCR detection method for the transgenic soybean ZH10-6 was established.The results showed that the method had great specificity.The optimal primer concentration(μmol/L)ratio of the 5 targets in the MPCR detection system(border A,border B,border C,border D,endogenous gene Lectin):0.8∶0.3∶0.1∶0.2∶0.05.The detection sensitivity of each target could reach 1%,which could be stably inherited among 3 generations,and was suitable for rapid and efficient detection of ZH10-6 insertion sites.The detection method established in this study improved the detection efficiency and reduced the detection cost.It provides a new detection technique for identification of the new variety of transgenic herbicide-tolerant soybean ZH10-6 transformant and its derivatives,which is technical support for its biosafety assessment,administrative supervision and intellectual property protection.

关 键 词：转基因耐除草剂大豆ZH10-6 多重PCR 分子特征 转化体特异性 检测方法 

分 类 号：S565.1[农业科学—作物学]