基于转录组测序筛选石蒜加兰他敏生物合成关键酶基因及功能鉴定

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.

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。

石蒜是一种重要的药用植物，因体内含有加兰他敏等石蒜科生物碱，而具有重要的潜在应用价值。本项目通过开展石蒜不同组织部位混合样品的全转录组和茉莉酸甲酯（MeJA）处理下差异转录组测序，分析完成了可能催化加兰他敏生物合成途径的候选氧甲基转移酶（OMT）和细胞色素P450酶（CYP）基因的筛选。在此基础上，结合基因克隆、底物化学合成、构建原核/真核表达体系、亚细胞定位、启动子克隆、拟南芥和石蒜转基因等一系列生理、生化及分子生物学方面的研究。重点对筛选得到的候选基因中的3个OMT基因（COMT、OMT和OMT-1）和1个CYP基因（CYP72A1）进行功能验证。研究结果表明：①COMT和OMT不具备催化加兰他敏生物合成中间产物-降孤挺花啶生成4’-氧甲基降孤挺花啶的作用；OMT-1则具有催化该反应的活性。不过，COMT在石蒜木质素合成途径中起到一定的作用。②通过克隆OMT-1基因的启动子序列，发现该基因启动子区含有受干旱、冷害等胁迫诱导及响应激素（MeJA和ABA）应答的重要顺式作用元件。③分别构建了OMT-1过表达和RNAi抑制载体，并通过转基因方法转入拟南芥和石蒜愈伤。④分析了石蒜内参基因的稳定性变化，以此为基础，对可能催化C-C酚耦合氧化反应的CYP71和CYP72基因家族表达量进行分析，发现其受不同胁迫和激素处理的诱导，且受诱导模式不同。⑤克隆得到与已知植物体内催化C-C酚耦合相似度较高的CYP72A1基因，并对其进行催化活性检测及功能验证；酵母真核表达系统获得的目标CYP72A1蛋白和烟草转基因瞬时表达植株提取蛋白可以催化底物4’-氧甲基降孤挺花啶生成新的化合物。本项目的实施，为进一步研究加兰他敏生物合成途径提供重要的基础，并为利用合成生物学对加兰他敏的从头（酪氨酸和苯丙氨酸）合成提供了理论基础；同时为工业化生产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。此外，还为深入研究石蒜科生物碱化合物的代谢调控提供了借鉴。