本项目拟在前期研究的基础上，以石蒜为材料，采用转录组测序技术筛选加兰他敏合成途径中的关键酶P450、COMT的候选基因；同时构建携带各候选基因的酵母工程菌株系，通过底物喂养及产物测定，筛选并验证目标基因。完成目标基因表达特性分析，鉴定其表达谱，亚细胞定位其编码的蛋白，克隆目标基因的启动子并进行序列分析；以部分缺失启动子融合GUS基因转拟南芥的方法验证其功能；采用酵母单杂交方法挖掘与启动子特异顺式作用元件结合的转录因子，克隆到转录因子基因并转拟南芥进行功能验证。将目标基因正义过表达载体和RNAi载体转入石蒜愈伤细胞，验证目标基因是否在加兰他敏合成途径中起作用；并将目标基因的正义过表达载体和RNAi载体转入拟南芥，进一步验证其生物学功能。通过以上各方面研究筛选并鉴定加兰他敏合成途径中的关键酶P450和COMT基因，揭示其调控机理，为加兰他敏生物合成研究和低成本生产加兰他敏提供理论和技术依据。

The goal of this program is to screen and identify the genes of two important enzymes (P450 and COMT) in biosynthesis of galanthamine, and reveal the molecular mechanism of their regulation in Lycoris radiata. For this purpose, the full-lenth cDNA of all P450 and COMT candidate genes would be presumed and cloned based on deep sequencing of the whole Lycoris radiata transcriptome by the next-generation sequenceing instrument and bioinformatics analysis. The target genes of P450 and COMT would be screened out and validated from the engineered yeast strains carrying genes of P450 and COMT, by detecting the products in the culture of engineered yeast with feeding specific substrate; and then the overexpression and RNAi vectors of the target genes would be constructed and transformated into Arabidopsis thaliana for the evaluation of the gene's biological functions. For further analysing the targeted gene's expression and regulation ,we would also carry out following studies: the expression profiles of the target P450 and COMT genes; the subcellular localization of proteins encoded by the target genes; cloning of promoters of P450 COMT and validation of their functions by transforming the vector with different deletion promoter fragement + GUS into Arabidopsis thaliana; identification of the transcription factor binding cis-acting elements in the promoters of target P450 and COMT by yeast one-hybrid; the function validation of the transcription factor gene by transformated Arabidopsis thaliana; and identification whether the target genes play a key role in the biosnythesis of galanthamine by transforming the overexpression and RNAi vectors of target genes of P450 and COMT into the callus cells of Lycoris radiata through Agrobacterium-mediated method. These studies could provide an important theoretical and technical basis for galanthamine's biosynthesis research and low-cost production.