多价抗虫水稻外源Bt基因的多引物多重PCR检测方法

Multi-primer Multiplex PCR Detection Method for the Exogenous Multivalent Insect-resistant Bt Genes in Transgenic Rice

旨在研究多价Bt抗虫转基因水稻的简便有效的检测方法。根据3个Bt转基因事件的插入位置,设计引物,通过多重引物PCR技术,利用9条引物得到6条大小存在明显差异的条带,同时检测3个Bt基因的转基因情况。水稻3个Bt抗虫基因(cry1Ab/Ac、cry2A、cry1C)分别来源TT51-1、T2A-1和T1C-19,其中代表TT51-1事件插入的阳性、阴性条带大小分别为937 bp、718 bp;代表T2A-1事件插入的阳性、阴性条带大小分别为600 bp、434 bp;代表T1C-19事件插入的阳性、阴性条带大小分别为495 bp、792 bp;通过琼脂糖凝胶电泳能快速对转Bt基因插入事件水稻的纯合、杂合、阴性进行检测。该技术克服了多重引物之间可能存在的引物间的相互干扰的技术难度,对不同的PCR试剂有很好的兼容性,可配合水稻DNA的快速提取,迅速对大量选育后代进行分析。该方法的建立为利用多价Bt基因进行抗螟虫水稻分子育种提供了一个简便高效的基因检测技术,有助于提高Bt基因检测效率,加快抗螟虫水稻的育种进程。

This work aims to explore a simple and effective method for detecting multivalent insect-resistant Bt genes in transgenic rice.Based on the insertion positions of 3 Bt genes(cry1Ab/Ac,cry2,and cry1C)from events(TT51-1,T2A-1,and T1C-19),9 primers were designed and used to obtain 6 target bands in significant differences by multiplex primer PCR technique.Subsequently,the positive and negative bands representing the TT51-1 event were 937 bp and 718 bp,those representing the T2A-1 event were 600 bp and 434 bp,and those representing the T1C-19 event were 495 bp and 792 bp.The detection of homozygous,heterozygous and negative Bt transgenic rice plants was rapidly conducted by agarose gel electrophoresis.This technology overcame the technical difficulties such as mutual binding interference among the multiple primers in the PCR process and presented fine compatibility with different PCR reagents.It can cooperate with rapid DNA extraction of rice to rapidly analyze a large number of breeding offspring.In conclusion,the establishment of this method provides a simple and efficient gene detection technology for rice stem borer-resistant molecular breeding with multivalent Bt gene,which is beneficial to increase the detection efficiency of Bt genes and to accelerate the breeding process of rice stem borer-resistant transgenic rice.