转基因大豆OsDREB3品系特异性定性PCR检测方法的建立

Establishment of event specific qualitative PCR for detecting transgenic soybean OsDREB3

为建立转基因大豆OsDREB3品系特异性定性检测方法,以转基因大豆OsDREB3为研究对象,通过染色体步行方法,成功获得转基因大豆OsDREB3上pCAMBIA1301质粒外源基因插入位点的5′端旁侧序列,其扩增片段覆盖了转化载体及转基因大豆基因组旁侧序列,扩增片段大小为344bp。同时根据旁侧序列设计引物,建立OsDREB3品系特异性定性PCR方法,以典型的转基因作物证明该方法检测转基因大豆OsDREB3具有高特异性,灵敏度为0.1%。

By chromosome walking PCR technique,the 5＇flanking sequences of the exogenous integration in the genome of in the pCAMBIA1301 plasmid of transgenic soybean OsDREB3 were cloned,and sequenced with the specific nested primers based on the border. According to the flanking sequence, the event specific primers were designed to amplify the fragment,which could span the exogenous DNA and carnation genome, and length was 344 bp. The event specific qualitative PCR detection method for transgenic soybean OsDREB3 was established. This assay was applied to detect typical genetically transgenic soybean OsDREB3, with high specificity, and the limit of detection exhibited 0. 1%. The results showed that the qualitative PCR assay was accurate, rapid and efficient for detection of transgenic soybean OsDREB3.