石蒜加兰他敏生物合成关键N甲基转移酶基因的鉴定及功能研究

Galanthamine is used for the treatment of Alzheimer’s disease most frequently in the world. The chemical synthesis of galantamine is very difficult, and the content of galanthamine in Lycoris radita is very low. So it is the most important to identify the functional genes in the biosynthesis route of galanthamine for the improvement of Lycoris resource. A N methyltransferase, catalyzing the N–demethylgalanthamine to galanthamine is used for the treatment of Alzheimer’s disease most frequently in the world. The chemical synthesis of galantamine is very difficult, and the content of galanthamine in Lycoris radita is very low. So it is the most important to identify the functional genes in the biosynthesis route of galanthamine for the improvement of Lycoris resource. A N methyltransferase, catalyzing the N–demethylgalanthamine to galanthamine at the last step, is still unknown, which is one of the key enzymes in the route. So the N methyltransferase gene would be identified and analyzed in this project. As it was found that MJ can induce the galanthamine accumulation remarkably in the previous study, transcript profiles of Lycoris aurea between MJ and CK combinded with the RNA de novo seq of Lycoris aurea were analyzed, and 9 sequences were selected as the candidates, the Unigen8814 among those was deduced as the aimed N methyltransferase. And then the aimed N methyltransferase gene in galanthamine synthesis would be identified by virus induced gene silencing (VIGS), and would be homologous cloned from Lycoris radiata. The identity and specific activity of the N methyltransferase from Lycoris radiata would be confirmed by heterologous expression of the protein in Escherichia coli and Pichia pastoris. Meanwhile the significant differences between wild and transgenic Lycroris radiata overexpressing or silencing the N methyltransferase gene by CRISPR/Cas9 would be observed in the aspects of gene expression and galanthamine accumulation. In addition, the promoter of the N methyltransferase gene would be cloned and the cis-elements would be analyzed and confirmed. These results would clarify the functions of the N methyltransferase gene, catalyzing the last step in galanthamine synthesis, and provide some theoretic basis for the improvement of Lycoris radiata by engineering.

加兰他敏是治疗老年痴呆症的优势药物，化学合成困难，在药源植物石蒜中含量极低，解析其合成路径中功能基因具有重要的理论和现实意义。而催化其合成终反应的N甲基转移酶基因尚未有报道。为了分离鉴定该基因，申请人基于茉莉酸甲酯（MJ）能显著诱导加兰他敏合成积累的发现，开展了忽地笑MJ处理的差异转录组测序，结合项目组已有的忽地笑RNA de novo数据库，筛选获得9条候选序列，其中Unigen8814极可能为目标基因。基于此，拟进一步通过病毒介导的基因沉默（VIGS）筛选，并同源克隆石蒜候选基因；进而通过原核、真核表达系统验证其蛋白催化特性；通过构建过表达和基于CRISPR/Cas9的基因敲除载体，转化石蒜，分析其在加兰他敏合成中的作用；并通过启动子调控元件的预测和缺失来分析其调控加兰他敏合成的特性。研究结果将全面解析催化加兰他敏合成终反应的N甲基转移酶的功能特性，为石蒜改良提供重要的理论和应用基础。

加兰他敏是临床上与治疗老年痴呆症的优势药物，石蒜属植物是其重要的来源。本项目在前期转录组测序的基础上，进一步完成石蒜不同组织部位的转录组测序，并结合加兰他敏在石蒜各组织部位的积累，综合分析了候选LrNMT基因。通过PCR克隆的方法获得3个LrNMT基因（LrNMT1，LrNMT2，LrNMT3）的全长cDNA；通过构建其原核/真核表达体系、亚细胞定位和拟南芥和转基因进行功能验证。研究结果表明：1）LrNMT基因在石蒜的不同组织部位中均有表达，呈现出组织特异性；LrNMT1，LrNMT2和LrNMT3基因受MeJA基因抑制表达； 2）由于体外表达的LrNMT重组蛋白以包涵体形式存在，未能检测其催化活性；3）亚细胞定位结果表明：LrNMT2定位在细胞质，而LrNMT1和LrNMT3主要定位在细胞质中的未知细胞器；4）LrNMT1基因含有多个内含子，利用CRISPR/Cas9原理设计并构建了基因编辑的敲除载体，同时还构建了35S:LrNMT1的过表达载体，并进行了石蒜转基因，目前分别获得了2个和4个敲除和过表达的幼芽；5）构建了LrNMT3基因过表达和反义的转基因载体，并进行拟南芥转基因，其中，反向过表达（抑制）LrNMT3基因促进拟南芥侧根数目的增加，并增强植株对NaCl和ABA的敏感性。本项目的实施，初步验证了候选LrNMT基因的功能，为加兰他敏的合成生物学研究奠定了一定的理论基础。