药用植物大戟二萜类生物合成中细胞色素P450研究

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。

“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.