野三七原人参三醇环氧酶（PE）基因克隆与功能分析

Ginsenosides exhibit a wide range of biological activities among plant species. Ginsenosides can be classified into four groups by the skeleton of their aglycones, namely oleanane-, protopanaxadiol-, protopanaxatriol- and ocotillol-type. Panax vienamensis var. fuscidiscus particularly and highly accumulates ocotillol-type Majonoside R2（MR2）, but its biosynthesis and regulation mechanism is still unclear, especially on the key reaction of cyclization of protopanaxatriol, which is catalyzed by protopanaxatriol epoxidase (PE). To elucidate the biosynthesis pathway of MR2 in P. vienamensis var. fuscidiscus, the gene of key enzyme (PE) will be cloned by the methods of transcriptome sequencing and rapid-amplification of cDNA ends(RACE). The catalytic characteristics of the enzyme and the function of PE gene will be detected by heterologous expression in yeast expression system and RNA interference using Agrobacterium rhizogenes-mediated genetic transformation. Furthermore, the temporal and spatial expression of PE gene and the effects of environment factors on their expression will be detected by semi-quantitative RT-PCR and Real-time PCR. The results of this study will provide a full understanding of biosynthesis of ocotillol-type ginsenoside, and explore the expression patterns of PE and the effects of environmental factors.

人参皂苷是人参属植物的主要活性成分，分为齐墩果烷型皂苷、原人参皂苷二醇、原人参皂苷三醇和奥克梯隆型四种皂苷。野三七（P. vienamensis var. fuscidiscus）具有高含量的奥克梯隆型皂苷,但奥克梯隆型皂苷的生物合成途径尚不明确，特别是其关键反应即原人参三醇环氧酶（PE）的催化原人参皂苷三醇的环化。本项目拟采用转录组测序和RACE技术克隆野三七PE基因，酵母表达和RNA干涉验证基因功能；并利用半定量RT-PCR和Real-time PCR方法检测PE基因的时空表达模式，希望能够解析奥克梯隆型珠子参皂苷MR2完整的生物合成途径，明确野三七PE的表达模式以及环境因子对其表达的影响。

野三七（P. vienamensis var. fuscidiscus）具有高含量的奥克梯隆型皂苷,但奥克梯隆型皂苷的生物合成途径尚不明确，特别是其关键反应即原人参三醇环氧酶（PvfSE）的催化原人参皂苷三醇的环化。本项目在转录组测序的基础上，克隆了PvfSE1、PvfSE2、PvfSE3全长序列，并登陆了NCBI数据库，登录号分别是KJ946467，KJ946468‏，KJ946469，补充了国内中药材的转录组数据。qRT-PCR分析发现PvfSE1、PvfSE2、PvfSE3在不同部位均有表达，但表达量不同，且对MeJA处理的反应也不同。PvfSE1在茎中表达量高，而PvfSE2和PvfSE3在叶片中表达量最高。.原核表达分析PvfSE1、PvfSE2、PvfSE3重组蛋白特性，分离纯化了表达蛋白，这表明三个基因可在体外正常表达，并构建了PvfSE1、PvfSE2、PvfSE3的植物表达载体和DNA敲除载体，为后续基因的转化和功能的验证奠定了基础。同时对野三七再生体系的建立进行了探索，确立了外植体的灭菌方式，但由于毛状根再生体系的建立进展较缓慢，后续基因功能的验证工作还在继续进行中。