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当前位置: 首页 > 文章 > 利用微滴数字PCR技术分析转基因大豆‘GE-J12’中外源基因的拷贝数 中国农业大学学报 2022,27 (1) 58-66
Position: Home > Articles > Analysis of the copy number of exogenous gene in transgenic soybean‘GE-J12'with droplet digital PCR Journal of China Agricultural University 2022,27 (1) 58-66

利用微滴数字PCR技术分析转基因大豆‘GE-J12’中外源基因的拷贝数

作  者：
赵新;刘双;刘娜;李瑞环;曹英芳;兰青阔;王永
单  位：
天津市农业科学院生物技术研究所;河北农业大学食品科技学院
关键词：
拷贝数;微滴数字PCR;转基因大豆;外源基因
摘  要：
为鉴定抗除草剂转基因大豆新品种‘GE-J12’的外源基因插入拷贝数,以该转化体的外源插入目的基因G2-EPSPS和GAT的序列、3′转化体特异性序列为靶序列,设计PCR扩增引物和TaqMan探针,并对引物探针特异性进行鉴定,同时以大豆内标基因Lectin为参照,建立微滴数字PCR拷贝数检测体系。特异性试验结果显示,只有以抗除草剂转基因大豆‘GE-J12’基因组DNA为模板才有扩增信号。以单株转基因大豆‘GE-J12’基因组DNA为模板,进行外源目的基因G2-EPSPS和GAT、3′转化体特异性序列的微滴数字PCR检测,转基因大豆‘GE-J12’的外源目的基因G2-EPSPS和GAT在基因组上的插入拷贝数均值分别为0.99和1.01。同时3′转化体特异性序列的拷贝数均值为1.00,验证单株转基因大豆‘GE-J12’为纯合子,因此鉴定该单株转基因大豆‘GE-J12’的外源基因在大豆基因组上为单拷贝插入,同时与Southern blot方法进行比较,结果一致。
译  名：
Analysis of the copy number of exogenous gene in transgenic soybean‘GE-J12'with droplet digital PCR
作  者：
ZHAO Xin;LIU Shuang;LIU Na;LI Ruihuan;CAO Yingfang;LAN Qingkuo;WANG Yong;Biotechnology Research Institute,Tianjin Academy of Agricultural Sciences;College of Food Science and Technology,Hebei Agricultural University;
关键词：
copy number;;droplet digital PCR;;transgenic soybean;;exogenous gene
摘  要：
In order to identify the copy number of exogenous gene of the new herbicide-resistant transgenic soybean‘GE-J12',droplet digital PCR(ddPCR)detection method was established.Using gene Lectin as soybean reference gene,and the sequences of the exogenous targeted genes G2-EPSPS and GAT,as well as the 3′event-specific sequences,the primers for PCR amplification and TaqMan probe were optimally designed.Specifically assay verified that only the genomic DNA template of transgenic soybean‘GE-J12'showed positive signals in the specificity testing.ddPCR analysis showed that the average copy numbers of G2-EPSPS and GAT were respectively 0.99 and 1.01 by using‘GE-J12'genomic DNA as template.Meanwhile,the copy number was determined as 1.00 for the 3′-end eventspecific sequence,and the new herbicide-resistant transgenic soybean ‘GE-J12'was confirmed as homozygous carrying one single transgenic insertion.The mentioned compare with Southern blot method,the results are consistent.

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