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本研究通过RNAi技术,使用发卡RNA结构沉默罂粟可待因酮还原酶(COR)与小檗碱桥酶(BBE)基因的表达。采用RT-PCR技术克隆得到COR和BBE基因全序列,同源性比较结果显示,它们与GenBank中已报道的COR和BBE基因高度同源;按照RNAi设计原则,筛选出了COR和BBE基因靶标序列,并采用重叠PCR法将其拼接成643 bp的融合基因,以中间载体pHANNIBAL和植物表达载体pCEPSPS为基础,构建ihpRNA植物表达载体,用农杆菌介导法将目的基因转入烟草中,共获得转基因植株78株,通过PCR检测从中筛选出了17株阳性植株,可初步确定目的基因已经整合到烟草基因组中。
In this study, we used RNAi technology to silence the expression of codeine-reductase (COR) and berberine-bridge enzyme (BBE) genes in poppy by hairpin RNA. The complete sequences of COR and BBE genes were cloned by RT-PCR. The homology results showed that they were highly homologous to the reported COR and BBE genes in GenBank. According to the RNAi design principle, the target sequences of COR and BBE genes were screened out , And was cloned into a 643 bp fusion gene by overlapping PCR. Based on the intermediate vector pHANNIBAL and the plant expression vector pCEPSPS, the ihpRNA plant expression vector was constructed, and the target gene was transferred into tobacco using Agrobacterium-mediated method. Totally 78 transgenic plants were screened out by PCR. Seventeen positive plants were screened out, which confirmed that the target gene had been integrated into the genome of tobacco.