三萜皂苷合成生物学元件的初步开发:三七鲨烯环氧酶编码基因克隆及表达模式分析

Development of the devices for synthetic biology of triterpene saponins at an early stage: cloning and expression profiling of squalene epoxidase genes in Panax notoginseng

中药合成生物学是将合成生物学思路引入中药天然产物次生代谢途径研究的一门新兴学科,其发展初期的首要任务是进行元件的开发与标准化,建立标准化的合成生物学元件库。三七(Panax notoginseng)作为人参属重要的药用植物,具有较高的药用价值,其主要活性成分是三萜皂苷。鲨烯环氧酶(squalene epoxidase)被认为是三萜皂苷与植物甾醇次生代谢途径中的关键限速酶,是三萜皂苷合成生物学研究的重要元件之一。本研究克隆获得三七中两种类型的鲨烯环氧酶编码基因(PnSE1、PnSE2),对其进行生物信息学分析,并通过实时荧光定量PCR(real-time PCR)方法检测其在4年生三七不同组织部位的表达模式,以及受茉莉酸甲酯(MeJA)诱导处理后基因的表达量变化,为实现中药合成生物学元件挖掘与开发奠定基础。PnSE1基因与PnSE2基因预测编码蛋白分别包含537和545个氨基酸,序列相似性为79%,但N端(前70个氨基酸)序列不保守。PnSE1基因在根、茎、叶、花中均有表达,以花中表达丰度最高;PnSE2基因只在花中表达量显著,其余组织较弱。PnSE1基因响应MeJA诱导,诱导24 h后在叶中的表达量最大;相同诱导条件下,PnSE2基因未响应诱导,表达水平无显著变化。结果表明,PnSE1和PnSE2基因具有不同的表达模式,在三七次生代谢产物合成中起不同催化作用,推测PnSE1基因参与三萜皂苷合成途径,PnSE2基因则在甾醇合成途径中起催化作用。

Synthetic biology of traditional Chinese medicine （TCM） is a new and developing subject based on the research of secondary metabolite biosynthesis for nature products. The early development of synthetic biology focused on the screening and modification of parts or devices, and establishment of standardized device libraries. Panax notoginseng （Burk.） F.H.Chen is one of the most famous medicinal plants in Panax species. Triterpene saponins have important pharmacological activities in P. notoginseng. Squalene epoxidase （SE） has been considered as a key rate-limiting enzyme in biosynthetic pathways of triterpene saponins and phytosterols. SE acts as one of necessary devices for biosynthesis of triterpene saponins and phytosterols in vitro via synthetic biology approach. Here we cloned two genes encoding squalene epoxidase （PnSE1 and PnSE2） and analyzed the predict amino acid sequences by bioinformatic analysis. Further, we detected the gene expression profiling in different organs and the expression level of SEs in leaves elicited by methyl jasmonate （MeJA） treatment in 4-year-old P. notoginseng using real-time quantitative PCR （real-time PCR）. The study will provide a foundation for discovery and modification of devices in previous research by TCM synthetic biology. PnSE1 and PnSE2 encoded predicted proteins of 537 and 545 amino acids, respectively. Two amino acid sequences predicted from PnSEs shared strong similarity （79%）, but were highly divergent in N-terminal regions （the first 70 amino acids）. The genes expression profiling detected by real-time PCR, PnSE1 mRNA abundantly accumulated in all organs, especially in flower. PnSE2 was only weakly expressed and preferentially in flower. MeJA treatment enhanced the accumulation of PnSE1 mRNA expression level in leaves, while there is no obvious enhancement of PnSE2 in same condition. Results indicated that the gene expressions of PnSE1 and PnSE2 were differently transcribed in four organs, and two PnSEs differently responded to MeJA stimuli. It was strongly suggested that PnSEs play different roles in secondary metabolite biosynthesis in P. notoginseng. PnSE1 might be involved in triterpenoid biosynthesis and PnSE2 might be involved in phytosterol biosynthesis.