应用单引物PCR获得以Bar基因为锚定基因的转基因水稻侧翼序列

Cloning Flanking Sequence of Bar gene by Single-primer PCR in Transgenic Rice

确定外源基因在水稻基因组上的插入位置,是转基因水稻研究的重要内容之一。为了建立一种简单、快速、准确的获取外源基因在水稻基因组上插入位置侧翼序列的方法,结合前人TAIL-PCR引物设计的原则和经验,以转入水稻基因组中的Bar基因为锚定基因,设计了既能作为锚定引物又能作为随机引物使用的一系列单引物,应用这些单引物通过单引物PCR成功获得了外源基因在水稻基因组插入位点的侧翼序列。这种方法与目前较为常用的TAIL-PCR相比,简化了70%左右的试验步骤,节省了60%以上的试验时间,是一种简便、快捷的获得未知侧翼序列的方法。

The insertion position of exogenous genes in targeted plant genomes are usually identified by adapter ligationmediated polymerase chain reaction, thermal asymmetric interlaced PCR, and restriction site extension PCR in transgenic rice research. However, there are various limitations in these methods, such as complexity of designing primers, and use of timeconsuming and multiplestep procedures. The goal of the present study was to establish an easier, and a more rapid and accurate method for cloning flanking sequence using singleprimer PCR in transgenic rice. Unknown flanking genome sequences in transgenic rice were successfully cloned using the singleprimer PCR method established in this study, with the gene as the anchor gene. Our data demonstrated that the singleprimer PCR is a more rapid and accurate method, justifying its application widely in cloning flanking sequences in transgenic rice.