Xia Zhihui, Gao Lifen, Luo Yuehua, Deng Xiaojian, Li Shigui, Zhai Wenxue
College of Agriculture, Hainan University, Haikou 570228, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):605-10.
Polymerase chain reaction (PCR) is a simple, quick and highly sensitive method. However the accuracy of the conventional PCR assay was often affected by false positives and false negatives. In this study, a protocol competitive PCR was used to reduce the false results in screening for selectable marker-free (SMF) Xa2l transgenic rice plants. The competitive template of Xa21 was the endogenous Xa2l homologous sequence located on chromosome 11. The competitive template of the selectable marker gene, hygromycin phosphotransferase (hpt), was an additive DNA extracted from hpt transgenic Nipponbare (Oryza sativa L). Through competitive PCR analysis of transgenic T1 plants produced by double right border binary vector, false positive or false negative samples were effectively diminished, and genuine SMF Xa21 transgenic plants were obviously obtained. Comparing with the conventional non-competitive PCR, competitive PCR increased the accuracy for selecting SMF Xa21 transgenic plants. The results of bacterial blight (BB) resistance tests and hygromycin B resistance assay of SMF Xa21 transgenic plants testified the reliability of this method.
聚合酶链式反应（PCR）是一种简单、快速且高度灵敏的方法。然而，传统PCR检测的准确性常常受到假阳性和假阴性的影响。在本研究中，采用了一种竞争性PCR方案来减少筛选无选择标记（SMF）Xa21转基因水稻植株时的错误结果。Xa21的竞争性模板是位于第11号染色体上的内源性Xa21同源序列。选择标记基因潮霉素磷酸转移酶（hpt）的竞争性模板是从hpt转基因日本晴（Oryza sativa L）中提取的附加DNA。通过对双右边界二元载体产生的转基因T1植株进行竞争性PCR分析，有效减少了假阳性或假阴性样本，并明显获得了真正的SMF Xa21转基因植株。与传统的非竞争性PCR相比，竞争性PCR提高了筛选SMF Xa21转基因植株的准确性。SMF Xa21转基因植株的白叶枯病（BB）抗性测试和潮霉素B抗性分析结果证实了该方法的可靠性。
