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

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.

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。

石蒜科植物体内中间代谢物4’-氧甲基降孤挺花啶的C-C酚耦合反应，是决定产生哪种重要类型的石蒜科生物碱的核心步骤。本项目利用忽地笑和中国石蒜转录组、全长转录组和代谢组联合分析，筛选到石蒜科生物碱生物合成途径中可能催化C-C酚耦合反应的关键CYP450s和桥酶（BBE）候选基因，并进行了功能研究。主要结果如下：（1）完成了忽地笑不同组织部位生物碱含量差异分析及中国石蒜不同组织部位代谢组测定和分析。（2）完成了忽地笑不同组织部位及茉莉酸甲酯（MeJA）处理下的差异转录组测序和数据分析；混合忽地笑不同组织部位及MeJA处理下的幼苗RNA，完成了PacBio SMRT全长转录组测序及数据分析；同时完成了中国石蒜不同组织部位的转录组测序及数据分析。在此基础上，进行转录组和代谢组数据联合分析，筛选获得了可能参与C-C酚耦合催化反应的CYP450s酶和BBE候选基因。（3）完成了CYP450候选基因LaCYP96T1的克隆及生物信息学分析，结合基于基因融合策略的CYP450及CPR筛选及活性验证体系，体外重组表达LaCYP96T1-ATR1蛋白，并对其进行了底物4’-氧甲基降孤挺花啶催化活性的初步验证，明确了LaCYP96T1具有催化C-C酚耦合反应的能力。通过定量PCR等手段对LaCYP96T1基因表达模式进行分析和验证；克隆得到忽地笑桥酶基因LaBBE1和LaBBE2，并对其序列及基因表达模式进行了分析，并通过原核表达初步鉴定了LaBBE2具有催化C-C酚耦合反应的活性，且与LaCYP96T1催化的反应类型不同。（5）构建了LaCYP96T1过表达和CRISPR/Cas9基因编辑载体，尝试和探索了不同条件下的石蒜愈伤转基因试验，获得了阳性愈伤；同时获得了过表达LaCYP96T1 的转基因拟南芥。本项目的实施，为后续深入研究并明确LaCYP96T1和LaBBE2基因在石蒜科生物碱生物合成中的作用奠定了一定的理论基础。

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