篩選影響日日春長春鹼含量之調控因子

Abstract

日日春（Catharanthus roseus）是一種具有藥用價值的植物，能合成多種植物次級代謝物，其中包括雙吲哚類生物鹼——長春新鹼（vinblastine）與長春花鹼（vincristine），兩者具有強效的抗癌活性，並廣泛應用於睪丸癌、乳癌與肺癌等疾病的臨床治療。然而，這些生物鹼在植物體內的天然產量極低，對藥品生產造成重大限制。 本研究旨在找出可能參與長春新鹼生合成途徑調控的轉錄因子（TF）。我們篩選了來自先前研究的245 個轉錄因子病毒誘導基因沉默（VIGS）品系。整體篩選流程分為三個階段。首先進行第一輪篩選，利用半定量 RT-PCR 檢測四個與長春新鹼生合成相關的標誌基因（TDC、STR、D4H 和 PRX1）的表現變化。接著進行第二輪篩選，使用即時定量 PCR（RT-qPCR）進一步驗證這些基因的表現變化。最後進行第三輪篩選，以質譜儀分析選定品系中萜吲哚生物鹼（TIA）的實際累積量。 在第一輪篩選中，我們發現69 個 VIGS 品系對標誌基因表現有明顯影響；其中有17 個品系在第二輪篩選中表現出顯著差異。我們從中挑選8 個品系進行代謝物分析，結果發現MYB-34與MYB-36 的沉默顯著改變了 TIA 的累積量。另一方面，作為正對照的MYC2與ORCA3在沉默後，無論是標誌基因表現或 TIA 含量皆未出現顯著變化，與預期結果不符。 總結而言，雖然本研究所建立的篩選系統仍有優化空間，但已成功提供 TIA 生合成調控網絡的嶄新線索，並找出具有潛力的新穎調控因子，可供未來深入探討。

Catharanthus roseus is a medicinal plant known for producing a variety of phytochemicals, including the bisindole alkaloids vinblastine and vincristine, which exhibit potent anticancer activities and are clinically used in the treatment of testicular, breast, and lung cancers. However, the natural yield of these alkaloids in C. roseus is extremely low, posing a major challenge for pharmaceutical production. In this study, we aimed to identify transcription factors (TFs) that may regulate the vinblastine biosynthesis pathway by screening 245 TF virus-induced gene silencing (VIGS) lines generated from previous studies. The screening process consisted of three stages. In the primary screening, we used semi-quantitative RT-PCR to evaluate the expression of four marker genes—TDC, STR, D4H, and PRX1—involved in vinblastine biosynthesis. The secondary screening was conducted using real-time quantitative PCR (RT-qPCR) to validate gene expression changes. In the third stage, we employed mass spectrometry to quantify the accumulation of terpenoid indole alkaloids (TIAs) in the selected VIGS lines. From the primary screen, we identified 69 candidate TF VIGS lines showing altered expression of marker genes. Of these, 17 lines exhibited significant fold changes in the secondary screen. Eight of these lines were selected for metabolite analysis, and we found that silencing of MYB-34 and MYB-36 significantly altered TIA accumulation. Conversely, silencing of MYC2 and ORCA3, which served as positive controls, did not result in significant changes in marker gene expression or TIA levels, contrary to our expectations. Overall, while the screening system still requires further optimization, this study provides new insights into the transcriptional regulation of TIA biosynthesis and identifies potential TF candidates involved in regulating vinblastine production.