主動誘變轉殖系牛樟芝活性成分分析與其分子層面之研究

Molecular studies on activation-tagged Antrodia cinnamomea and analysis of its active compounds

陳建甫(Chien-fu Chen)

指導教授 ： 蔡新聲 ； 陳靖棻

朝陽科技大學/理工學院/生物技術與化學工程研究所碩士班/碩士(2008年)

摘要

牛樟芝，學名為Antrodia cinnamomea，為台灣特有之藥用真菌，具抗癌、抗發炎和調節免疫等療效。本試驗為了提高牛樟芝菌絲體之多醣及三萜類等活性成分，以農桿菌品系EHA105及主動誘變載體pTAG-8進行牛樟芝菌絲體基因轉殖並找出受影響之基因，試驗成功獲得16個轉殖系菌絲體(A∼P)，經由南方墨點法分析後得知有81.25％的轉殖系菌絲體為單一T-DNA嵌入，其中不僅菌絲顏色發生變化，且所有轉殖系之生長速度皆慢於未轉殖菌絲體(20％∼40％)。利用酚硫酸法進行胞外多醣含量測定，其中顯著高於未轉殖菌絲體的轉殖系為A、C、K、L、N與O。以HPLC測定同時存在牛樟芝子實體與菌絲體中之dehydrosulphurenic acid與dehydroeburicoic acid等三萜類，得知具治療攝護腺癌效果的前者在轉殖系D、H、I、J與K中的含量明顯高於未轉殖菌絲體；具抗發炎效果的後者則於轉殖系A、B、D、H與I中含量較高。同時含高量dehydrosulphurenic acid與dehydroeburicoic acid之轉殖系H的清除DPPH自由基能力為所有轉殖系中最佳，相當於子實體。在經由inverse PCR選殖得知其T-DNA嵌入基因為DIMETHYLALLYL TRYPTOPHAN SYNTHASE後，確定該基因表現量在轉殖系H中受到抑制，因此推測該基因應與轉殖系H含高量dehydrosulphurenic acid與dehydroeburicoic acid相關。
關鍵字: 牛樟芝、農桿菌、主動誘變

關鍵字

主動誘變 ； 農桿菌 ； 牛樟芝

並列摘要

Antrodia cinnamomea is well known in Taiwan as a source of traditional Chinese medicine for the treatment of cancer and inflammation. In order to increase the content of active compounds, such as polysaccharides and triterpenoids in mycelia, Agrobacterium tumefaciens strain EHA105 harboring a standard binary vector pTAG-8 was used to generate the transgenic mycelia population. Sixteen transgenic mycelia (A~P) were obtained and confirmed by Southern blots(81.25％ of them with single copy insertion). Changes in mycelia color and growth rate were observed in all transgenic lines. Exopoly- ssacharides contents in transgenic lines A, C, K, L, N and O were significantly increased. Triterpenoids like dehydrosulphurenic acid (used for prostate cancer therapy) and dehydroeburicoic acid (used for anti-inflammation) in both fruiting bodies and mycelia were remarkably increased in transgenic lines D, H, I, J, K and A, B, D, H, I, respectively. Transgenic line H, containing high dehydrosulphurenic acid and dehydroeburicoic acid, was observed to have high scavenging activity. In transgenic line H, the DIMETHYLALLYL TRYPTOPHAN SYNTHASE(DTS) was as confirmed by gene expression. Mutation in DTS might cause accumulation of dehydrosulphurenic acid and dehydroeburicoic acid in transgenic line H.
Key words: Antrodia cinnamomea, Agrobacterium tumefaciens, Activation
tagging.

並列關鍵字

Antrodia cinnamomea ； Agrobacterium tumefaciens ； Activation tagging

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