罂粟COR和BBE基因RNAi载体构建及烟草转化  

Construction and tobacco transformation of COR and BBE genes hairpin RNA vector of Papaver somniferum

作　　者：陆艳梅[1,2] 张金文[1,2] 魏玉洁[3] 魏贵民[1,2] 张艳红 高宜峰[1,2] 

机构地区：[1]甘肃省作物遗传改良与种质创新重点实验室,甘肃兰州730070 [2]甘肃农业大学农学院,甘肃兰州730070 [3]甘肃省农垦农业研究院,甘肃兰州730070 [4]甘肃省中医学院,甘肃兰州730070

出　　处：《药学学报》2013年第7期1169-1177,共9页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(31160303);教育部博士学科点研究基金项目(2007733008);甘肃省卫生厅重点中医药科技项目(2008HDTC02)

摘　　要：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。The gene expressions of codeinone reductase （COR） and berberine bridge enzyme （BBE） in Papaver somniferum were blocked by RNA hairpin of RNA interference （RNAi）. The complete sequences of COR and BBE genes were cloned by reverse transcription-polymerase chain reaction （RT-PCR）, the results of homology comparison revealed that the cloned COR and BBE genes had high homology with the other gene family members reported in the GenBank. The target sequences of COR and BBE genes were screened in accordance with the design principle of RNAi, a 643 bp fusion gene was obtained by the method of overlapping PCR, then plant expression vector ihpRNA was constructed based on intermediate vector pHANNIBAL and plant expression vector pCEPSPS. With that 78 transgenic plants were obtained through Agrobacterium-mediated and 17 positive plants were screened by PCR, that could initially indicate that the target fragments of COR and BBE gene had been integrated into tobacco genome.

关 键 词：罂粟 COR和BBE基因 RNAI 转化 

分 类 号：S336[农业科学—作物遗传育种]