药用植物大戟二萜类生物合成中细胞色素P450研究

“Jing Daji”, the root of Euphorbia pekinensis Rupr, is a common medicine in the Folk. The research suggested that Euphorbia pekinensis have double physiological efficiency that its products possess antibiosis, antivirus and anti-tumor activity as well as strong toxicity. This double properties is closely related to structural type of diterpenes in Euphorbia pekinensis. The cytochrome P450 are involved in the biosynthesis of diterpenes, thus it will be of great importance in clarifying the cytochrome P450 involved in biosynthesis of diterpenes in Euphorbia pekinensis. It has been reported that salicylic acid can promote the accumulation of diterpenes in suspension cell of Euphorbia pekinensis. In this research, the transcriptome of salicylic acid treated and untreated suspension cell of Euphorbia pekinensis will be sequenced. Combined with fluorescence quantitative RT-PCR and high resolution mass spectrometry technology, the data of sequencing will be analyzed and the responded genes of cytochrom P450 enzyme in the pathway of diterpene metabolism will be obtained. The RACE and genome walking technology will be used to amplify these P450 genes and which will be done deep analysis on structure and function. At the same time the new CRISPR/Cas9 technology will be used to knock out these genes to conform the function in biosynthesis of diterpenes. These research will lay the foundation and provide scientific basis for developing secondary metabolism engineering in improving the content of active component and reduce toxicity in Euphorbia pekinensis.

药用植物大戟的根京大戟是一种民间常用药材，研究表明其有双重生理活性，既有抗菌，抗病毒和抗肿瘤等生理活性，又有致毒作用，这种双重生理活性与其所含有的二萜结构类型密切相关，细胞色素P450(CYP450)参与了萜类多样性结构的形成，因此弄清大戟二萜类生物合成中参与的CYP450具有重要意义。本项目在已有研究表明水杨酸促进大戟悬浮细胞二萜类化合物积累的基础上，拟应用最新转录组手段，结合荧光定量RT-PCR和高分辨质谱等技术，比较分析水杨酸诱导前后CYP450基因表达与二萜类积累的相关性。采用RACE和基因步移技术获得二萜合成相关CYP450基因全序列，深入分析这些CYP450的结构与功能。同时利用最新的CRISPR/cas9基因敲除技术确认这些CYP450基因在二萜类生物合成中的作用。研究结果为大戟降低毒性提高活性成分的次生代谢工程奠定基础和提供理论依据。

Casbane烷型二萜类化合物是药用植物大戟中主要的活性成分，目前已经从大戟根中分离鉴定到多种这类活性化合物。近年来对二萜基本骨架形成已经有了突破性的进展，但是对casbane烷型二萜生物合成途径的研究并不深入。casbane烷型二萜由蓖麻烯合成酶催化GGPP合成蓖麻烯，然后在CYP450及其他相关酶的作用下生成结构各异的casbane烷型二萜。因此研究大戟中casbane烷型二萜合成相关的CYP450基因可了解各casbane烷型二萜的生物合成途径。通过本项目的研究，获得了一些参与casbane烷型二萜生物合成的CYP450基因。项目主要研究结果包括：1）建立了大戟根、茎、叶和愈伤的转录组数据库，基于大戟活性二萜成分组要存在于根中，挖掘casbane烷型二萜生物合成相关的CYP450基因；2）水杨酸诱导可提高大戟愈伤中的活性二萜成分含量，因此对水杨酸处理和未处理的大戟愈伤进行转录组测序，获得有响应的CYP450基因；3）通过生物信息软件对这些CYP450基因进行预测分析。同时通过PCR扩增这些基因，并构建酵母表达载体，与蓖麻烯合成酶基因一起转入酵母验证基因的功能；4）通过基因过表达/基因编辑等方法，将CYP450基因转化到大戟的愈伤组织中，通过测定蓖麻烯及casbane烷型二萜的含量检测这些基因的功能。通过本项目的研究，阐释了大戟casbane烷型二萜合成相关的CYP450基因，为我国药用植物天然产物的开发和利用提供了理论基础。