东方泽泻中倍半萜合酶的功能表征及产物鉴定

Functional characterization and product identification of sesquiterpene synthases in Alisma orientale (Sam.) Juz.

摘要:

中药泽泻为泽泻科泽泻属多年生水生或沼生草本植物东方泽泻[Alisma orientale (Sam.) Juzep.]或泽泻(Alisma plantago-aquatica Linn.)的干燥块茎，是常用利水渗湿药，其富含萜类成分，尤以倍半萜类化合物为重要活性物质，具有抗癌、抗炎等多种药理作用。截至目前，东方泽泻倍半萜生物合成通路的研究较为匮乏，本研究旨在解析泽泻倍半萜的生物合成通路，挖掘并鉴定参与倍半萜合成的关键功能基因，为阐明泽泻药效物质基础及后续质量标志物筛选与利用提供理论依据。因此，采用转录组学和分子生物学手段，挖掘并克隆出9个具有功能的东方泽泻倍半萜合酶基因AoTPSs，通过酵母异源表达体系，鉴定其生物化学功能，证实这些基因可催化合成β-榄香烯(β-elemene)、α-石竹烯(α-caryophyllene)、反式-橙花叔醇[(E)-nerolidol]、β-石竹烯(β- caryophyllene)、α-法尼烯(α-farnesene)等倍半萜类化合物。本研究为深入解析泽泻倍半萜的生物合成机制及资源开发奠定了基础。

Abstract:

The Chinese herbal medicine Rhizoma Alismatis is the dried tuber of Alisma orientale (Sam.) Juzep. or Alisma plantago-aquatica Linn., perennial aquatic or marsh herbaceous plants belonging to the family Alismataceae. It is a commonly used herb for inducing diuresis and eliminating dampness. It is rich in terpenoids, especially sesquiterpenoids as important active ingredients, which exhibit various pharmacological activities such as anticancer and anti-inflammatory effects. To date, studies on the sesquiterpenoid biosynthetic pathway in Alisma orientale (Sam.) Juzep. remain insufficient. This study aimed to elucidate the sesquiterpenoid biosynthetic pathway in Rhizoma Alismatis, identify and characterize key functional genes involved in sesquiterpenoid biosynthesis, and provide a theoretical basis for clarifying its pharmacodynamic material basis and the subsequent screening and application of quality markers. Therefore, employing approaches of transcriptomics and molecular biology, identified and cloned nine functional sesquiterpene synthase genes (AoTPSs) from A. orientale. Using a yeast heterologous expression system, we characterized their biochemical functions, confirming that these genes can catalyze the biosynthesis of sesquiterpenoids such as β-elemene, α-caryophyllene, (E)-nerolidol, β-caryophyllene, and α-farnesene. This study lays a foundation for in-depth elucidation of the biosynthetic mechanism and resource development of Alisma sesquiterpenoids.