Integrated construction and event-specific real-time PCR of transgenic rice Bt Shanyou 63 | 转基因水稻Bt汕优63的整合结构和品系特异性定量PCR方法

Chinese. 为更好地监测转基因抗虫水稻(Oryza sativa L.)Bt汕优63品系的存在，满足转基因标识管理的要求，同时也为了解决阳性植物基因组DNA不容易获得的问题，本研究采用热不对称交互PCR(TAIL-PCR)、Genome Walking和长链PCR(LD-PCR)方法测定了基因枪转化的转cry1Ab/cry1Ac融合基因水稻品系Bt汕优63的外源插入DNA全序列，构建了含有Bt汕优63 3'旁侧和水稻内标基因gos9序列的标准质粒分子pMDBt63作为标准物质，建立了Bt汕优63实时荧光定量PCR方法。外源插入DNA全长9 818 bp，位于水稻基因组10号染色体(AP008214)第5348630位，由8个来源于两个转化质粒的DNA片段组成，其中两个cry1Ab/cry1Ac表达框以正向串连的方式插入。gos9和3'旁侧两个定量标准曲线的相关系数(R2)分别为0.9997和0.9992，扩增效率(E)分别为98.05%和99.46%。每反应100 ng模板DNA的检测限(LOD)为10拷贝，定量限(LOQ)为100拷贝。此外，对已知5%和1%转基因含量的混合样品DNA进行定量检测，结果的平均值分别为4.98%和1.07%。结果表明标准质粒pMDBt63作为标准物质建立的定量方法可用于Bt汕优63的定量检测。

English. In order to execute the labeling regulations and solve the inadequate availability of positive plant genome DNA, the existence of insect resistant rice (Oryza sativa L.) Bt Shanyou 63 has to be monitored, In this study, the complete sequence of foreign DNA insertion of transgenic cry1Ab/cry1Ac fusing gene rice line Bt Shanyou 63 transformed by particle bombardment method was determined using thermal asymmetric interlaced PCR (TAIL-PCR), Genome Walking and long distance PCR (LD-PCR). The Real-time PCR assay was established using the standard plasmid pMDBt63 containing 3' flanking sequence of Bt Shanyou 63 and rice endogenous gos9 as the reference material. The length of the complete sequence of foreign DNA was 9 818 bp, located at position 5348630 of chromosome 10 (AP008214) in rice genome and composed of eight recombinant fragments including two expression boxes of cry1Ab/cry1Ac fused gene at tail to head direction. The square regression coefficients (R2) of the two standard curves for gos9 and 3' flanking sequence were 0.9997 and 0.9992 and the efficiencies (E) were 98.05% and 99.46%, respectively. The detection limit (LOD) was 10 copies and the quantitative limit (LOQ) was 100 copies in 100 ng of DNA template for one reaction. In addition, two mixed samples with known Bt Shanyou 63 contents (5% and 1%) were quantified using the established Real-time PCR and the mean values were 4.98% and 1.07%, respectively. The above results showed that the established method employing pMDBt63 as the reference material can be used reliably for Bt Shanyou 63 quatification.