转基因耐逆大豆FvC5SD-L05事件的T-DNA 侧翼序列分析及其特异性检测
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1.吉林省农业科学院 农业生物技术研究所;2.东北农业大学

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吉林省农业科学院基本科研经费杰出青年项目(KYJF2021JQ001)


T-DNA flanking sequence analysis and specific detection of transgenic soybean FvC5SD-L05
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Affiliation:

1.Institute of Agricultural Biotechnology,Jilin Academy of Agricultural Sciences;2.Northeast Agricultural University,Harbin

Fund Project:

The Basic Research Funds of JAAS (KYJF2021JQ001)

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    摘要:

    转FvC5SD-L05基因大豆事件是利用农杆菌介导的大豆子叶节遗传转化法将从金针菇克隆到的FvC5SD基因导入大豆栽培品种沈农9号中获得的纯合耐旱转基因大豆株系,该材料具有良好的耐旱特性和农艺性状。为了明确该份转基因大豆材料的分子特征及其检测方法,从而推进其生物安全性评价工作,本研究以转基因大豆FvC5SD-L05的T4代纯合株系为研究对象,利用Southern杂交方法鉴定了外源基因的拷贝数;及基因组重测序技术分析外源基因FvC5SD在大豆基因组中插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转FvC5SD基因耐逆大豆L05事件的特异性定性PCR检测方法。该方法特异性强、灵敏度高,能够在FvC5SD-L05基因组DNA含量为0.1%的模板中检测出转基因成份,实现了对转FvC5SD基因耐逆大豆L05事件的特异性检测,进一步为该份转基因大豆及其衍生产品的检测和身份识别提供技术依据。

    Abstract:

    The FvC5SD gene cloned from Flammulina velutipes and introduced into soybean cultivar Shennong 9 by Agrobacterium mediated genetic transformation of soybean cotyledon nodes, and the transgenic soybean FvC5SD-L05 events showed good drought tolerance and agronomic properties. to clarify the molecular characteristics and detection methods of the transgenic soybean FvC5SD-L05 material for its biosafety evaluation, we analyzed the copy numbers of FvC5SD gene by Southern hybridization and identified the position and direction of the insertion site of foreign gene in soybean genome, basing the 4th generation of FvC5SD-L05 events. Meanwhile, by PCR amplifying the left and right flanking sequences of exogenous T-DNA, we also established a specific qualitative PCR detection method for FvC5SD-L05 by its left and right flanking sequences. This method has strong specificity and high sensitivity. It can detect transgenic components in the template with FvC5SD-L05 genomic DNA content of 0.1% and realize the specific detection of FvC5SD-L05 event of stress resistant soybean with FvC5SD gene, which further provides technical basis for the detection and identification of transgenic soybean and its derivatives.

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  • 收稿日期:2022-04-03
  • 最后修改日期:2022-04-18
  • 录用日期:2022-05-06
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