基于重测序鉴定耐盐转基因大豆事件外源T-DNA整合位点及特异性检测

Identification of T-DNA insertion and specific detection of transgenic soybean event based on re-sequencing technology

[目的]转基因大豆事件L8014是本研究前期利用农杆菌介导的大豆子叶节高效遗传转化技术体系,将来源于山菠菜的基因BADH导入大豆(Glycine max)‘Williams 82’而获得具有良好耐盐(1.5%NaCl)性状的转基因大豆(该材料已经完成环境释放试验)。T-DNA插入位点侧翼序列的获得与分析对于转基因事件的安全评价及应用非常重要,本文旨在明确该转基因大豆材料的分子特征及其检测方法,以进一步推进安全评价。[方法]利用Southern杂交检测转基因大豆外源基因的拷贝数;基于基因组重测序技术,采用BWA(Burrows-Wheeler-Alignment)方法将重测序数据与T-DNA序列以及大豆基因组序列进行比对分析。[结果]T-DNA在大豆基因组10号染色体上的基因组区间3925017—3926022位点,插入方式为反向插入;结合PCR扩增技术获得外源T-DNA整合位点的左、右边界侧翼序列,基于该序列建立了转BADH基因大豆事件L8014的特异性PCR检测方法。[结论]本研究获得了转基因事件的整合位点及侧翼序列,方法快速、结果可靠,为转基因大豆及其衍生产品的检测提供了技术支撑。

[Objectives]In the early stage of this study,we introduced gene BADH into the soybean cultivar‘Williams 82’to generate the transgenic event L8014,which showed good salt tolerance(1.5%NaCl)and had been cleared for environmental release.Obtaining and analyzing the flanking sequence of T-DNA insertion site were very important for the safety evaluation and application of transgenic events.The characteristics of the transgenic soybean L8014 material were clarified to promote its biosafety evaluation.[Methods]In this study,we detected the copy number of BADH gene by Southern hybridization.Based on genome resequencing technology,Burrows-Wheeler-Alignment(BWA)method was used to compare and analyze the resequencing data with the T-DNA sequence and soybean genome sequence.[Results]T-DNA was found to be in nucleotides 3925017 to 3926022 in the chromosome 10 of soybean genome.Combined with PCR amplification technology,the left and right boundary flanking sequences of exogenous T-DNA integration site were obtained.[Conclusions]Based on these sequences,an event-specific PCR detection method was established,which might provide rapid and accurate detection to identify the transgenic soybean L8014.