LlOMT和LlNMT调控长筒石蒜中抗AD药物加兰他敏合成通路的机制研究

Galanthamine (Gal) is an isoquinoline alkaloid produced only by Amaryllidaceae family plants, which is used for the treatment of Alzheimer's disease. Lycoris longituba contains high amount of Gal, however, the commercial use of Gal is restricted by their limited availabilities of plant resources. In that case, engineering Gal production through the elucidation of its biosynthesis pathway became the hotspots in this field. The evidence suggests that methyltransferase LlOMT and LlNMT are the key enzymes in the biosynthesis of Gal, and their gene functions still await discovery. .In the previous study, two full length methyltransferase candidate genes were identified from Lycoris longituba through NGS and PacBio Sequel full-length RNA-Seq. It was found that Gal accumulation was positively correlated with the expression of candidate genes, suggesting that genes play important roles in the synthesis of Gal. Based on the previous study, this project will do the following works 1) the function of candidate genes will be verified through over expression of genes, RNA interference and in vitro enzyme activities. 2) the temporal and spatial accumulation of Gal will be clarified, and the molecular mechanism underlying Gal accumulation was revealed through gene expression pattern and SWATH-MS analysis. 3) homologous sequences of the target genes in different species will be cloned and the correlation between the difference of Gal content and the variation of the gene sequences will be studied. This results will explain the possible mechanism of LlOMT and LlNMT regulating Gal synthesis at molecular level, and will also provide candidate genes and targets for the improvement of Gal content by metabolic engineering.

加兰他敏（Gal）为石蒜科植物特有的临床治疗老年痴呆症（AD）的药物，长筒石蒜含有高含量的Gal，但植物资源有限使之应用受限，因此通过代谢工程来提高Gal含量前景广阔，证据表明甲基转移酶LlOMT和LlNMT是Gal生物合成的关键酶，其基因功能未见报道。.前期通过全长转录组测序，分离LlOMT和LlNMT基因的全长序列，并发现Gal积累与候选基因的表达呈正相关，暗示基因在Gal合成中起重要作用。本项目将在前期基础上，1）通过基因的过量和抑制表达、体外酶活分析，验证候选基因功能。2）研究Gal时空积累规律，通过基因表达模式和SWATH-MS蛋白组学差异分析，揭示目的蛋白调控Gal积累的分子机理。3）克隆不同种资源中基因的同源序列，揭示种间Gal含量差异与基因序列变异的相关性。本结果将从分子水平解析LlOMT和LlNMT调控Gal合成的机理，将为利用代谢工程提高Gal的含量提供候选基因和靶点。

加兰他敏（Gal）为石蒜科植物特有的临床治疗老年痴呆症（AD）的药物，长筒石蒜含有高含量的Gal，但植物资源有限使之应用受限，因此通过代谢工程来提高Gal含量前景广阔，证据表明甲基转移酶LlOMT和LlNMT是Gal生物合成的关键酶，其基因功能未见报道。本项目前期通过全长转录组测序，结合不同器官的转录组测序，分离LlOMT和LlNMT基因的全长序列，并发现Gal积累与候选基因的表达呈正相关，暗示基因在Gal合成中起重要作用。后续通过基因的表达分析、亚细胞定位和体外酶活分析，验证候选基因功能。LlOMT可以催化norbelladine成4'-O-methylnorbelladine，过表达LlOMT能够提高加兰他敏含量。LlNMT具有的N-甲基转移酶活性，催化产物中检测到少量Gal。对长筒石蒜中LlOMT和LlNMT时空表达和Gal积累的相关性进行分析，Gal含量与两个基因的表达呈相似的规律。对不同生物碱含量的长筒石蒜等代表样品进行SWATH-MS定量蛋白组学分析，共定量到2193个蛋白，不同组样品间蛋白定量信息存在明显差异，碳代谢和氨基酸合成是两个变化最大的通路，生物碱通路蛋白也存在显著变化，Gal合成途径的关键基因序列在不同样品间存在差异，这些可能是不同样品间Gal差异性积累的影响因素。本结果将从分子水平解析LlOMT和LlNMT调控Gal合成的机理，将为利用代谢工程提高Gal的含量提供候选基因和靶点。