Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

中文翻译：

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在水仙属植物中参与阿尔茨海默病药物加兰他敏生物合成的去甲甘草碱 4'-O-甲基转移酶的克隆和表征。阿夫。假水仙。

加兰他敏是一种石蒜科生物碱，用于治疗阿尔茨海默病的症状。该化合物主要从水仙花 (Narcissus spp.)、雪花莲 (Galanthus spp.) 和夏季雪花 (Leucojum aestivum) 中分离出来。尽管它作为一种药物很重要，但尚未鉴定出与加兰他敏生物合成途径有关的基因。缺乏生物合成途径的遗传信息是许多重要植物药物合成生物学平台开发的一个限制因素。信息的缺乏主要是由于在非模式生物中寻找生化途径酶和基因的传统方法的局限性。使用最近几项技术改进的新生物信息学方法被应用于在提议的加兰他敏生物合成途径中搜索基因，由于蛋白质中的强特征氨基酸序列，首先靶向甲基转移酶。使用 Illumina 测序，构建了水仙花的从头转录组组装。BLAST 用于鉴定在该转录组中包含植物 O-甲基转移酶特征的序列。然后使用 HAYSTACK 程序来鉴定适合叶、球茎和花序组织中加兰他敏生物合成模型的甲基转移酶。确定了在提议的加兰他敏生物合成途径中将去甲甘草定甲基化为 4'-O-甲基去甲泡兰定的一种候选基因。该甲基转移酶 cDNA 在大肠杆菌中表达，并通过亲和层析纯化蛋白质。发现所得蛋白质是拟定的加兰他敏生物合成途径的去甲贝拉定 4'-O-甲基转移酶 (NpN4OMT)。