抗病转基因大豆事件B4J8049外源T-DNA整合位点分析及特异性检测
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Q93

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国家转基因生物新品种培育科技重大专项(2016ZX08004-004);吉林省农业科学院创新工程项目(C6215000220、C7208000307)


Analysis of the Foreign T-DNA Insertion Site and Event-Specific Detection of the Disease-Resistant Transgenic Soybean B4J8049
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    摘要:

    大豆疫霉根腐病是由卵菌纲病原菌—疫霉菌(Phytophthora sojae)引起的大豆毁灭性病害之一。本研究前期利用农杆菌介导转化技术,将广谱抗病基因hrpZm导入栽培大豆Williams 82,获得高抗疫霉根腐病转基因大豆新品系B4J8049,并已进入环境释放试验阶段。为进一步推进该转化事件的生物安全评价及应用,本研究采用热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)方法分析外源T-DNA整合位点右边界序列,并依据其序列特征,建立该转化事件特异性检测方法。Southern杂交检测结果表明,转基因大豆事件B4J8049外源T-DNA插入拷贝数为1个。根据外源T-DNA插入片段序列设计3条嵌套特异性检测引物,与简并引物组合进行TAIL-PCR扩增反应,获得外源TDNA插入位点右边界序列。BLAST分析(https://soybase.org/)表明,外源T-DNA片段以单拷贝形式反向插入的方式整合到大豆基因组中第8号染色体的187 824位点。在此基础上,依据插入位点右边界序列设计检测引物,建立转基因大豆事件B4J8049特异性检测方法。本研究为该抗病转基因事件B4J8049及其衍生产品特异性检测提供依据。

    Abstract:

    Phytophthora root and stem rot, caused by an oomycete pathogen Phytophthora sojae, is one of the most devastating diseases in soybean production. In our previous study, we introduced a broad-spectrum disease resistant gene hrpZm into the soybean cultivar ’Williams 82’by the Agrobacterium-mediated transformation to generate the transgenic event B4 J8049,which exhibited high resistance to P. sojae and approved for environmental release. To advance safety assessment and application of the transgenic soybean event, we analyzed the insertion site of the foreign T-DNA in the genome of the event B4 J8049 using the thermal asymmetric interlaced PCR(TAIL-PCR), and established the event-specific detection method based on the flanking sequence at the right border of the T-DNA insertion. Southern blot analysis confirmed the single-copy insertion of the foreign T-DNA in the soybean genome.Three nested specific primers based on the T-DNA sequence and the arbitrary degenerate primers were designed to amplify the right border flanking sequence by TAIL-PCR. BLAST analysis(https://soybase.org/) revealed that the foreign T-DNA was inserted into the position 187 824 on the soybean chromosome 8 in the reverse way. Based on the flanking sequence of the right border, an event-specific detection method was established for the transgenic soy-bean B4 J8049 and its derivative products.

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包婉莹,仲晓芳,杜茜,赵倩倩,杨向东.抗病转基因大豆事件B4J8049外源T-DNA整合位点分析及特异性检测[J].东北农业科学,2018,43(5):21-26.

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  • 收稿日期:2018-06-24
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  • 在线发布日期: 2024-12-06
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