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PCR是一种简单、迅速、灵敏的检测方法,但假阳性与假阴性却影响了它在常规应用中的准确性。本研究利用竞争性PCR解决无标记Xa21转基因水稻PCR检测中的假阳性与假阴性问题。标记基因潮霉素基因(Hygromycin phosphotransferase,hpt)的竞争模板是外加的日本晴hpt转基因植株基因组DNA,抗白叶枯病基因Xa21的竞争模板是待测水稻内源的位于第11染色体上的Xa21同源基因序列。利用这一方法对双右边界T-DNA载体转化产生的转基因T1代植株进行分析,可以有效地减少或排除假阳性或假阴性样品,选出真正的转基因阳性植株。与常规PCR相比竞争性PCR提高了无标记Xa21转基因植株筛选的准确性。对获得的无标记Xa21转基因植株进行白叶枯抗病鉴定与潮霉素抗性鉴定证实了该方法的可靠性。
PCR is a simple, rapid and sensitive detection method, but its false positive and false negative affect its accuracy in routine applications. In this study, competitive PCR was used to solve the false positive and false negative PCR detection in unlabeled Xa21 transgenic rice. The competitive template for the marker gene hygromycin phosphotransferase (hpt) was genomic DNA supplemented with Nipponbare hpt transgenic plants. The competition template for the Xb21 gene was rice endogenous Xa21 on chromosome 11 Source gene sequence. Using this method to analyze transgenic T1 generation plants transformed by double right border T-DNA vector can effectively reduce or eliminate false positive or false negative samples and select true transgenic positive plants. Competitive PCR increases the accuracy of screening of unlabeled Xa21 transgenic plants compared to conventional PCR. The obtained marker-free Xa21 transgenic plants were identified as blight resistance and hygromycin resistance. The reliability of the method was verified.