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| 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 |
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赵新1,刘双1,刘娜1,李瑞环1,曹英芳2,兰青阔1,王永1*
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| (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) |
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| 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。 |
| 关键词: 拷贝数 微滴数字PCR 转基因大豆 外源基因 |
| DOI：10.11841/j.issn.1007-4333.2022.01.06 |
| 投稿时间：2021-01-06 |
| 基金项目:国家科技重大专项(2019ZX08004001-005) |
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| Analysis of the copy number of exogenous gene in transgenic soybean ‘GE-J12' with droplet digital PCR |
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ZHAO Xin1,LIU Shuang1,LIU Na1,LI Ruihuan1,CAO Yingfang2,LAN Qingkuo1,WANG Yong1*
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| (1.Biotechnology Research Institute, Tianjin Academy of Agricultural Sciences, Tianjin 300384, China;2.College of Food Science and Technology, Hebei Agricultural University, Baoding 071001, China) |
| Abstract: |
| 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 event-specific 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. |
| Key words: copy number droplet digital PCR transgenic soybean exogenous gene |
