土壤磷素高效利用转基因大豆特异性PCR检测方法

Event-specific qualitative and quantitative PCR detection methods for high phosphate-efficient transgenic soybean

华南农业大学根系生物学研究中心采用拟南芥的紫色酸性磷酸酶基因AtPAP15转化大豆品系粤春03-3(YC03-3),获得了酸性磷酸酶活性明显提高、可高效利用土壤磷素的转基因大豆新品系AP15-1。本研究以AP15-1为研究对象,应用TAIL-PCR技术,根据载体序列设计特异引物,获得了转化载体左侧插入的旁邻序列。设计事件特异性检测引物,进行PCR扩增,只能在AP15-1的样品中扩增出特异性条带,进一步用实时荧光定量PCR作分析,结果显示,该引物对重复性好,融解曲线显示只有一个特异峰值。本实验应用该引物对建立的检测方法,检测的灵敏度可以达到0.01%,实时荧光定量PCR检测的极限值可以达到9个基因组的拷贝数,能够满足对转基因大豆新品系AP15-1及其衍生品种检测的需要。

An event-specific transgenic detection method was established for previously developed transgenic soybean line AP15-1,in which AtPAP15 was introduced into elite soybean variety Yuechun 03-3（YC03-3） to improving the ATPase activity and thus significantly improving phosphate efficiency.The left border（LB） of integration junction sequence was 566bp in length cloned by TAIL-PCR（thermal asymmetric interlaced PCR）.Sequencing result indicated that the 566bp fragment including 308bp from vector and 258bp from soybean genome.A pair of event-specific primers was designed according to the junction sequences to amplify a 119bp fragment with PCR.Quantitative real-time PCR detection assays were consequently conducted.The detection sensitivity reached 0.01% in qualitative PCR,and the detective limit for quantitative PCR assay reached 9 copies in soybean genome.Thus a method by event-specific PCR was successfully developed to identify and quantify AP15-1 and its derived lines.