台灣蟲草交配基因座、代謝產物和抗癌活性特性分析

Characterization of mating loci, metabolites and anti-cancer activity of Ophiocordyceps formosana

王瑛璟(Ying-Jing Wang)

指導教授 ： 沈湯龍

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703792

摘要

冬蟲夏草(簡稱蟲草)富含多種二次代謝物，包括蟲草(cordycepin)、蟲草多醣、蟲草酸及超氧化歧化脢等，現其研究指出具抗癌、抗氧化、降血糖、免疫調節等諸多功能。其中，蟲草素與腺苷(adenosine)結構相似，抗制癌細胞效果也較佳。了解其交配相關功能性基因將有利於以人工方式培養蟲草子實體並且獲取更多有效成分及應用。正如同冬蟲夏草為昆蟲寄生菌，2013年夏季本實驗室從桃園縣拉拉山採集到受感染的蟲體，成功分離出臺灣蟲草(Ophiocordycpes formosana) 菌株，並於沙氏葡萄糖與酵母培養基(SDAY medium)培養後以菌絲體型態保存。一般認為真菌子實體中蟲草素含量較高，因子實體為有性世代構造，需交配基因座(mating-type loci)中的MAT1-1與MAT1-2兩種性別的參與。故透過使用專一性引子進行聚合酶連鎖反應，分別增幅菌株的交配基因座中MAT1-1-1的α box及MAT1-2-1的high-mobility group (HMG)，可判斷菌株性別。進而將含有MAT1-1與MAT1-2的菌絲培養於白米培養基，可成功培養出臺灣蟲草的子實體。另外，使用高壓液相層析法（high pressure liquid chromatography, HPLC）分析菌絲體粗萃物，發現其腺苷含量較豐，蟲草素較低。經MTS assay測試，高濃度的菌絲體萃取物可有效抑制人類淋巴瘤細胞HL60增殖。利用高壓液相層析儀與質譜儀偵測處理後的人類淋巴瘤細胞株U937的裂解產物，可發現蟲草素存在，故認定腺苷及蟲草素含量可能為影響細胞存活率主因。利用二次質譜分析細胞中物質的結構，可應用於探討細胞代謝過程的變化和鑑定相關化合物。未來可用於探討台灣蟲草子實體具有抗癌活性的二次代謝物，並建立二次代謝物、抗癌活性與交配基因座之間的關係，以提供更多蟲草於癌症治療上的有效資訊。

關鍵字

臺灣蟲草 ； 子實體 ； 萃取物 ； 交配基因座 ； 抗癌活性

並列摘要

Dong Chong Xia Cao (or Chong Cao) is rich in bioactive secondary metabolites, e.g. cordycepin, cordycepic acid, and polysaccharides attributing to many medicinal uses. For instance, cordycepin is an adenosine antagonist to be effective on anti-cancer and anti-viral activities. As an entomopathogen, we successfully collected and isolated an epi-typified Ophiocordyceps formosana from mummified darkling beetle bodies in Lalashan in 2013. The mycelia are continued to cultivate and preserved in the Sabouraud’s dextrose agar supplemented with yeast extract (SDAY) medium. Noticeably, fruiting bodies possess a higher level of cordycepin than mycelia. In addition to an optimized light intensity, temperature, and nutrient, the opposite mating type, MAT1-1 and MAT1-2, are required for fruiting body development of O. formosana. We have identified the α box or the high-mobility group (HMG) on mating type loci to show the existence of differential mating types among isolates of O. formosana, and induced fruiting bodies formation by solid-state fermentation. On the other hand, high adenosine and low cordycepin content were detected in the crude extraction of O. formosana mycelia by high pressure liquid chromatography (HPLC) analysis. High concentration of mycelia extract was showed a good effect of inhibition of human leukemia HL60 cells proliferation by MTS assay. Also, the signal of cordycepin in cell lysate of U937 was detected by liquid chromatography tandem mass spectrometry (LC–MS). The networking of tandem mass spectra (MS/MS) was showed the library of treated-cell lysate and helped to identify the relative compound. However, the anti-cancer effects and bioactive metabolic derivatives are waiting for being confirmed and explored by purifying the crude extract. This study is anticipated to provide more alternative medicine for cancer treatments.

並列關鍵字

Ophiocordyceps formosana ； fruiting body ； extract ； mating-type loci ； anti-cancer effect

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