Food Inspection Center, Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China.
Acta Biochim Biophys Sin (Shanghai). 2013 May;45(5):416-21. doi: 10.1093/abbs/gmt016. Epub 2013 Feb 28.
In order to establish a specific identification method for genetically modified (GM) wheat, exogenous insert DNA and flanking sequence between exogenous fragment and recombinant chromosome of GM wheat B73-6-1 were successfully acquired by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. Newly acquired exogenous fragment covered the full-length sequence of transformed genes such as transformed plasmid and corresponding functional genes including marker uidA, herbicide-resistant bar, ubiquitin promoter, and high-molecular-weight gluten subunit. The flanking sequence between insert DNA revealed high similarity with Triticum turgidum A gene (GenBank: AY494981.1). A specific PCR detection method for GM wheat B73-6-1 was established on the basis of primers designed according to the flanking sequence. This specific PCR method was validated by GM wheat, GM corn, GM soybean, GM rice, and non-GM wheat. The specifically amplified target band was observed only in GM wheat B73-6-1. This method is of high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of GM wheat B73-6-1.
为建立转基因小麦的特异鉴定方法，利用常规 PCR 和热不对称交错 PCR 技术，成功地获得了转基因小麦 B73-6-1 中外源插入 DNA 及其与重组染色体侧翼序列。新获得的外源片段覆盖了转化基因的全长序列，如转化质粒和相应的功能基因，包括标记 uidA、除草剂抗性 bar、泛素启动子和高分子量谷蛋白亚基。插入 DNA 之间的侧翼序列与 Triticum turgidum A 基因（GenBank：AY494981.1）具有高度相似性。根据侧翼序列设计引物，建立了转基因小麦 B73-6-1 的特异 PCR 检测方法。该方法通过 GM 小麦、GM 玉米、GM 大豆、GM 水稻和非 GM 小麦进行了验证。仅在 GM 小麦 B73-6-1 中观察到特异扩增的目的条带。该方法对 GM 小麦 B73-6-1 的鉴定具有高度特异性、高重复性、快速鉴定和良好的准确性。
