石蒜科生物碱合成关键C-C酚耦合酶细胞色素P450s的分离与功能分析

石蒜科生物碱多具有重要药用价值，其植物体内核心代谢通路已明确，申请人团队克隆了其中多个合成酶基因并验证功能；然而，决定形成全部三种核心构型石蒜科生物碱的关键酶C-C酚耦合CYPs酶(P450s）仍有待鉴定。本申请拟在前期转录组测序基础上，利用石蒜属植物忽地笑不同部位石蒜科生物碱含量和转录本表达协同分析，筛选克隆候选CYPs基因。通过忽地笑P450功能性表达系统、分子模拟和定点突变等方法，鉴定获得分别具4’-氧甲基催化降孤挺花定对-对'、对-邻'和邻-对'位C-C酚耦合的CYPs，并明确其生化特性和催化机制；利用忽地笑过表达/基因敲除（CRISPR/Cas9系统）转基因技术，验证CYPs基因在忽地笑石蒜科生物碱合成中的功能；结合RNA-Seq、启动子克隆等解析CYP基因受诱导表达模式。本研究将为高含量石蒜科生物碱忽地笑品种培育提供依据，也为利用生物工程手段合成重要石蒜科生物碱提供理论基础。

The core metabolic biosynthesis pathway of the Amaryllidaceae alkaloids, which exhibit a wide range of bioactivities, such as antitumor, antiviral, antibacterial, antifungal, antimalarial and analgesic, have been illuminated clearly. Previously, some candidate genes involved in the biosynthesis of Amaryllidaceae alkaloids were cloned and analyzed by the applicant’s research team. However, the key enzymes (cytochrome P450, CYPs) which catalyze a C-C phenol-coupling reaction that can have para-para’, para-ortho’, or ortho-para’ regionspecificity have not been identified. In this project, based on the former transcriptome sequencing of Lycoris aurea, RNA-Seq regarding the different tissues of L. aurea including leaf, root, bulb, flower and stem will further be performed. Meanwhile, the distribution pattern and contents of Amaryllidaceae alkaloids and their intermediates will also be determined. The correlation between gene expression and Amaryllidaceae alkaloids will be analyzed and used to characterize the candidate CYP genes. In addition, by using the CYP functional expression system constructed from L. aurea cytochrome P450 reeducates, molecular simulation and site-directed mutagenesis, the biochemical characteristics and catalytic mechanism of CYP will be clarified. Moreover, by using the transgenic technology such as gene overexpression and CRISPR/Cas9 gene editing, the important role and function of CYP involved in Amaryllidaceae alkaloids biosynthesis will be illuminated. Meanwhile, the promoter sequence of CYP genes will be cloned and analyzed, and its core promoter region (CPR, including regulatory cis-elements), which is critical for the responsiveness of the gene to different stimuli, will also be identified. Combined with the RNA-Seq analysis, the inducible expression patterns of CYP genes will be characterized. In this study, the function of key C-C phenol-coupling CYP involved in the Amaryllidaceae alkaloid biosynthesis pathway of Lycoris aurea will be clarified. The results obtained here will provide the theoretical basis for the bioengineering of the important Amaryllidaceae alkaloids in the genus Lycoris, and contribute importantly to better understanding of the cultivation of Lycoris with high Amaryllidaceae alkaloids contents.