反向PCR克隆转基因水稻的外源基因旁侧序列

摘要

On the basis of inverse PCR, we have founded a IPCR technique to clone foreign gene's flanking sequence in transgenic rice, which is suitable to treat mass materials. We used mini-preparation protocol to extract total DNA of transgenic rice, and DNA was digested by more than 10 times restriction endonuclease overnight in 50μL. The digested fragments were purified and self-ligated in a reaction volume of 20 μL. In order to enhance the specificity of PCR amplifying flanking sequences, the nested-PCR was used, and hot-start PCR and touchdown PCR were combined with it. Thirty five of foreign gene's flanking sequences have been cloned in transgenic rice, the cloned fragments, sizes are 300～750 bp, and the specificity of PCR product has been proved by Southern blot. The result showed that this IPCR technique is quick, convenient and stable for flanking sequence cloning.%以反向PCR（IPCR）为基础建立了适合于处理大量材料的克隆转基因水稻中外源基因旁侧序列的技术体系。该方法中用小量法提取转基因水稻总DNA；总DNA用10倍过量的限制性内切酶在50 μL反应体积中进行过夜酶切；酶切片段在20 μL体积中进行自连接，之后进行套式PCR（nested-PCR）扩增旁侧序列。在套式PCR中结合了热启动PCR和降落PCR技术以增强PCR反应的特异性。用这种方法，本实验室在一周内克隆了35个转基因水稻株系中外源基因的旁侧序列，长度在300～750 bp之间，PCR产物的特异性用Southern杂交进行了证明。实验结果表明这个方法具有快速、稳定和高效的优点。