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  

Analysis of the copy number of exogenous gene in transgenic soybean‘GE-J12’with droplet digital PCR

作　　者：赵新[1] 刘双 刘娜[1] 李瑞环 曹英芳 兰青阔[1] 王永[1] ZHAO Xin;LIU Shuang;LIU Na;LI Ruihuan;CAO Yingfang;LAN Qingkuo;WANG Yong(Biotechnology Research Institute,Tianjin Academy of Agricultural Sciences,Tianjin 300384,China;College of Food Science and Technology,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]天津市农业科学院生物技术研究所,天津300384 [2]河北农业大学食品科技学院,河北保定071001

出　　处：《中国农业大学学报》2022年第1期58-66,共9页Journal of China Agricultural University

基　　金：国家科技重大专项(2019ZX08004001-005)。

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。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.

关 键 词：拷贝数 微滴数字PCR 转基因大豆 外源基因 

分 类 号：TS201.6[轻工技术与工程—食品科学]