大豆转化体E8A7027外源T-DNA分析及特异性检测体系建立

Analysis and Specific Detection System Establishment of T-DNA Insertions for Transgenic Soybean E8A7027

为进一步分析转AgGlpF基因耐盐转基因大豆E8A7027转化体的分子特征信息并对其进行特异性检测,本研究分离该转化体的旁侧序列并建立其特异性PCR检测体系。基于基因组重测序技术并结合Sanger测序技术,将基因组重测序分析获得的序列信息与参考大豆基因组进行对比,确定E8A7027转化体的T-DNA区在大豆基因组上的插入位点及其5′端和3′端的旁侧序列。根据旁侧序列设计PCR检测引物,并对检测体系的特异性、灵敏度进行实验室内验证。结果显示:转基因大豆E8A7027的整合位点为Chr09染色体的33886331位点,整合方式为单拷贝插入,受体基因组DNA在插入位点缺失了1段58 bp序列,分别获得E8A7027大豆的5′端旁侧序列910 bp和3′端旁侧序列802 bp。根据这两个旁侧序列设计引物建立的PCR检测方法,能够特异性地将E8A7027大豆转化体与其他转基因作物和非转基因大豆区分开,方法的检测灵敏度为0.05%。研究结果可为E8A7027转化体的安全评价和监管检测提供技术支撑。

To further analyze the molecular characteristics and detect the specificity of the transgenic soybean E8A7027 transformant with AgGlpF gene,the proposed study isolated the flanking sequence of transgenic soybean event E8A7027 and established a specific PCR detection system.Based on genome resequencing and Sanger sequencing,the sequence information obtained by genome resequencing analysis was aligned with the reference soybean genome to determine the insertion loci,the flanking sequences at 5′and 3′ends of T-DNA region.The PCR primers were designed according to the event-specific sequence,and the specificity and sensitivity of the PCR assay were vertified in the laboratory.The results confirmed that the integration site of transgenic soybean E8A7027 was Chr09:33886331 with a single-copy insertion.The recipient genome DNA missed a 58 bp sequence at the insertion loci.The 910 and 802 bp flanking sequences at 5′and 3′end of E8A7027 event were obtained,respectively.The event-specific PCR with primers designed based on the two flanking sequences was enable to specifically distinguish the GM soybean event E8A7027 from other GM crops and non-GM soybeans,the sensitivity of this assay reached 0.05%(w/w).The result can provide technical support for the safety evaluation and regulatory of E8A7027 transformants.