八角蓮的松脂醇-落葉松樹脂醇還原酶於菸草葉片與羅勒毛狀根表現之研究

Abstract

鬼臼素是由鬼臼屬植物中分離出具有抗病毒能力的一種木脂素，其衍生物etoposide為可以抑制癌細胞的有絲分裂而做為化療藥物使用。鬼臼素在天然資源中相當少見，因此很難滿足對其日益增長的需求，因此，如何大量生產鬼臼毒素及其衍生物的新方法是一個迫在眉睫的問題。羅勒是一種富含酚酸和萜類化合物的草本植物，迷迭香酸、咖啡酸和綠原酸是羅勒中含量最豐富的酚類化合物，在先前的研究中曾進行迷迭香酸合成酶的RNA干擾（RASi）以增加苯基類丙烷化合物前驅物的累積，鬼臼素生合成的第一步是透過松脂醇-落葉松樹脂醇還原酶 （PLR）將松脂醇轉化為secoisolariciresinol。本研究於煙草葉片中進行八角蓮PLR瞬時表達，以了解其在植物細胞中的表現位置及活性。 PLR可同時在表皮細胞的細胞質和細胞核中被觀察到。在體外測試中則未檢測到PLR的催化活性。接著，將PLR轉形或與RASi一起轉形進羅勒毛狀根以生產開環異落葉松樹脂醇。在這些毛狀根的代謝物分析中，僅在PLR與RASi毛狀根中發現濃度為0.008 ppm/毫克乾重的松脂醇，在其它毛狀根中未觀察到PLR的其他產物落葉松樹脂醇和secoisolariciresinol。接下來將測量羅勒毛狀根中PLR的RNA和蛋白質表現量，並進一步分析其的代謝體學，以強化PLR的生產效率。

Podophyllotoxin is a lignan isolated from Podophyllum species and has antivirus ability. The derivative, etoposide, is also used as a medication for chemotherapy to inhibit cancer cell mitosis. Podophyllotoxin is rare in the natural source so it is hard to satisfy the growing demand. A new strategy to produce podophyllotoxin and its derivatives is an urgent problem. Ocimum basilicum (basil) is a valuable herb that is rich in phenolic acids and terpenoids. Rosmarinic acid, caffeic acid, and chlorogenic acid are the most abundant phenolic compounds in basil. The RNA interference of rosmarinic acid synthase (RASi) was expressed in the previous study to enrich the phenylpropanoids precursor accumulation. The first step of podophyllotoxin biosynthesis is to convert pinoresinol into secoisolariciresinol by the enzyme pinoresinol-lariciresinol reductase (PLR). In this study, PLR from Podophyllum pleianthum transient expression in the leaves of Nicotiana benthamiana was conducted first to realize its subcellular localization and activity in plants. PLR was expressed in the cytoplasm and nucleus of epidermal cells simultaneously. However, the catalytic activity of PLR was not detected in the in vitro test. Then, PLR was transformed into the basil hairy roots individually or with the RASi construction to the synthesis of secoisolariciresinol. In the metabolic analysis of these hairy roots, only pinoresinol was found in the PLR with RASi hairy roots with a low concentration of 0.008 ppm/mg DW. Other products of PLR were not observed in the hairy roots. The RNA level and protein expression of PLR will be determined, and the metabolome of basil hairy root will be further analyzed in the next step to optimize PLR expression in hairy roots for podophyllotoxin biosynthesis.