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可待因酮还原酶(COR)与小檗碱桥酶(BBE)是吗啡合成代谢途径的关键酶,其活性大小直接影响着吗啡合成途径中生物碱的代谢合成。采用RT-PCR从罂粟幼叶克隆出COR和BBE基因全序列,同源性比较结果显示,它们与GenBank上已报道的COR和BBE基因高度同源。利用blast及分子生物学软件DNAStar对COR和BBE基因的cDNA序列同源性进行分析比较,分别从各基因中筛选和克隆了一段同源性极低、约400～500bp的片段;并应用重叠PCR法将其拼接成744bp的融合基因BC,以中间载体pHANNIBAL和植物表达载体pART27为基础,构建了以CaMV35S启动子驱动的含有“正向BC融合片段-pdk内含子-反向BC融合片段”的ihRNAi植物表达载体,通过转化野生罂粟,初步研究了以COR和BBE基因为靶标的RNAi对内源吗啡合成的抑制效果,为进一步培育低吗啡高蒂巴因的罂粟种质提供了依据。
Codeoxin reductase (COR) and berberine bridge enzyme (BBE) are the key enzymes in the morphine metabolic pathway, and their activity directly affects the metabolic synthesis of alkaloids in the morphine synthesis pathway. The complete sequences of COR and BBE genes were cloned from poppy young leaves by RT-PCR. Homology comparison showed that they were highly homologous to the reported COR and BBE genes in GenBank. Using DNAStar, a blast and molecular biology software, to analyze and compare the cDNA sequence homology of COR and BBE genes, a fragment of 400 ~ 500bp with very low homology was screened and cloned from each gene. Method to spliced into a 744bp fusion gene BC, based on the intermediate vector pHANNIBAL and plant expression vector pART27, constructed a CaMV35S promoter containing “positive BC fusion fragment-pdk intron - reverse BC fusion fragment ”Of the ihRNAi plant expression vector, through the transformation of wild poppy, the preliminary study of the COR and BBE gene-targeted RNAi endogenous morphine synthesis inhibitory effect, to provide a basis for further breeding of low morphine high opium poppy germplasm .