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研究生: 謝富貴
論文名稱: 新日本靈芝、環紋靈芝純培養菌絲體之生理性狀及核醣體DNA定序之研究
Cultural, Physiolocal and Ribosomal DNASequencing Studies ofGanoderma neojaponicumand G. zonatum
指導教授: 葉增勇
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2002
畢業學年度: 91
語文別: 中文
論文頁數: 78
中文關鍵詞: 靈芝新日本靈芝環紋靈芝純培養菌絲體核醣體DNA
英文關鍵詞: G. neojaponicum, G. zonatum, Ribosomal DNASequencing, pure cultures
論文種類: 學術論文
相關次數: 點閱:181下載:11
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  • 新日本靈芝(G. neojaponicum)為分佈於台灣、大陸及日本,寄主為針葉樹及竹類等,環紋靈芝(G. zonatum)為產於美國佛羅里達州和喬治亞洲,其所寄生之宿主侷限於椰子類(palms) 之樹木。本研究以台灣新日本靈芝及美國環紋靈芝為菌種,純培養其菌絲體以探討兩個地區的靈芝,其菌絲體的生理特性,並利用聚合酶連鎖反應法得到核醣體DNA之內轉錄區(ITSⅠ&ITSⅡ),將其核酸序列進行分析比較其相似性,以提供台灣新日本靈芝及美國環紋靈芝生物多樣性之進一步資訊。
    新日本靈芝及環紋靈芝的純培養性狀中,兩者菌褥(mycelial mat)正面皆為白色,背面呈現褐色,生殖菌絲(generative hyphae)皆具有扣子體(clamp connection)及出現腫大的細胞(swelling cell),皆能分泌細胞外氧化酵素,包括漆氧化酵素(laccase)及過氧化酵素(peroxidase),而兩者皆不能分泌酪胺酸酵素(tyrosinase),屬於木材白腐菌(white rotting fungus),能利用木材之木質素(lignin)、亞纖維素(hemicellulose)與纖維素(cellulose)。
    新日本靈芝在麥芽抽出物培養基MEA及馬鈴薯葡萄糖培養基PDA的最適生長溫度範圍皆為24~28℃,環紋靈芝在MEA及PDA培養基的最適生長溫度範圍為32℃,兩者在溫度16℃、36℃時,無論是以MEA培養基或PDA培養基培養時,生長速率皆明顯較差。酸鹼度在pH 2.4~6.1範圍內對新日本靈芝的生長趨勢無太大影響,而環紋靈芝在pH 4.6時生長狀況最好。新日本靈芝對葡萄糖濃度的基本需求約40g/L,但葡萄糖濃度超過80g/L時,新日本靈芝的菌絲乾重並不隨供給的碳源增加,葡萄糖濃度在40~80g/L時對環紋靈芝的生長最佳。新日本靈芝與環紋靈芝的菌絲體以澱粉培養的生長表現優於以葡萄糖或蔗糖培養。新日本靈芝對無機氮的濃度需求不高,低無機氮源濃度(0.02N)時生長狀況較佳,對有機氮源的濃度需求為0.04~0.08N,有機氮源濃度超過0.4N時,則會抑制菌絲的生長。環紋靈芝在低無機氮源濃度(0.04N)時菌絲生長最佳,無機氮源濃度超過0.08N時,對菌絲的則生長有不良的影響,而對有機氮源反應則為濃度增加,生長情況趨勢漸佳。綜合各項研究結果,新日本靈芝及環紋靈芝菌絲體的生長特性、生理性狀隨生存環境因素不同而有所差異。
    菌絲核醣體的DNA定序結果,新日本靈芝的ITSⅠ-5.8S-ITS Ⅱ核苷酸序列分別為195-158-201 bp。環紋靈芝的ITS-5.8S-ITS Ⅱ核苷酸序列分別為208-158-195 bp。環紋靈芝與新日本靈芝及G.formosanum 0109親源關係較近。研究結果顯示rDNA的核苷酸序列分析可以輔助瞭解靈芝種間分子的演化情形。

    Ganoderma neojaponicum was obtained from Taiwan, while G. zonatum was obtained from U. S. A. Hence their pure cultures were used for morphological, physiological studies and ribosomal DNA sequencing analysis.
    The morphological characters of G. neojaponicum and G. zonatum were both observed as the white color on the mycelial mat’s surface, brown color on the other side, clamped generative hyphae and the appearance of swelling cells in culture.
    The positive reaction in extracellular oxidases which included laccase and peroxidase, but negative in tyrosinase, indicated G. neojaponicum and G. zonatum belong to white rotting fungi, and showed that they can utilize lignin and cellulose of wood。
    The results of physiological tests were shown as follows:
    1.Optimum temperature for the mycelial growth of G. neojaponicum was at 24~28℃ on both of MEA and PDA, while G. zonatum was at 32℃ both on MEA and PDA.
    2. Optimum pH for the mycelial growth of G. zonatum was 4.6, while G. neojaponicum was not prominent.
    3. Glucose concentration at 40~80g/L was the optimum condition for the mycelial growth of G. neojaponicum and G. zonatum. The increasing glucose concentration neither increased nor decreased mycelial growth rate of G. neojaponicum, but decreased mycelial growth rate of the G. zonatum.
    4.Starch was the best carbon source for both mycelial growth of G. neojaponicum and G. zonatum.
    3. Optimum nitrogen concentration of ammonium nitrate (NH4NO3) was at 0.02N for the mycelial growth of the G. neojaponicum, but at 0.04N for the G. zonatum. Whereas the mycelial growth of G. zonatum was in proportion to the concentration of L-Asparagine (organic nitrogen form) , but not for G. neojaponicum.
    Sequencing analyses of the ITS1-5.8S-ITS2 ribosomal DNA in G. neojaponicum are 195bp-158bp-201bp and in G. zonatum are 208bp-158bp-195bp respectively. The sequences data showed that G. zonatum was close to G.formosanum 0109 and G. neojaponicum. The phylogenetic relationship tree constructed from their study will aid to realize the molecular evolution among Ganoderma spices.

    摘要………………………………………………………………1 英文摘要…………………………………………………………3 壹、緒論 一、靈芝之分類與研究簡史……………………………………5 二、靈芝屬之分佈………………………………………………6 三、靈芝屬之巨觀形態特徵……………………………………7 四、靈芝屬之微觀形態特徵……………………………………7 五、靈芝屬之生長發育…………………………………………8 六、靈芝屬之寄生及生態特性…………………………………10 七、靈芝屬之成分與其藥效……………………………………11 八、靈芝純培養菌絲體之生理性狀……………………………12 九、有關靈芝屬核醣體DNA序列研究……………………….12 十、研究目的……………………………………………………15 貳、 材料與方法 一、菌種來源……………………………………………………16 二、純培養菌絲體之生長特性觀察 1.純培養菌絲體之生長特性觀察…………………………….16 2. 測定有無細胞外氧化酵素……………………..………….16 3. 測定細胞外氧化酵素之種類…….………………………17 三、菌絲體對不同環境條件反應試驗 1. 溫度生長試驗……………………………………………..18 2. 酸鹼度試驗………………………………………………..18 四、菌絲體的生理試驗 1.葡萄糖濃度試驗…………………………………………...19 2.不同碳源試驗……………………………………….……..20 3.無機氮源濃度試驗…………………………………………20 4.有機氮源濃度試驗………………………………………...20 五、菌絲體之核醣體DNA序列定序 1.萃取菌絲體基因體DNA………………. …………………21 2.聚合酶連鎖反應系統……..……………………………….22 3.聚合酶連鎖反應產物之檢測…………………….………..24 4.自動雷射螢光去氧核醣核酸定序儀………………………24 5.核酸序列分析………………………………………………24 參、結果 一、純培養菌絲體之性狀觀察……………………………….26 二、菌絲體對不同環境條件反應試驗 1. 溫度生長試驗…………………..………………………..26 2. 酸鹼度試驗………………………………………………27 三、菌絲體生理反應試驗 1. 葡萄糖濃度試驗………………………………………....27 2. 不同碳源試驗…………………………………………....28 3. 無機氮源濃度試驗………………………………………28 4. 有機氮源濃度試驗………………………………………29 四、菌絲體之核醣體DNA序列定序……………………..29 肆、討論……………………………………………………….30 伍、參考文獻………………………………………………….38 表目錄 表一 rDNA內轉錄區的相關位置圖及轉錄rDNA所需之引子………….49 表二 細胞外氧化酵素之測定結果……………………………………54 表三 新日本靈芝(G. neojaponicum CCRC36049) rDNA核苷酸序列65 表四 環紋靈芝(G. zonatum Yeh FL-05) rDNA核苷酸序列.………66 表五 新日本靈芝(G. neojaponicum CCRC36049)與已發表之新日本靈芝(G. neojaponicum gi: 537986) ITS Ⅱ核苷酸序列之比較…….67 表六 環紋靈芝(G. zonatum Yeh FL-05)與已發表之環紋靈芝 (G. zonatum gi: 5764506) ITSⅠ與ITS Ⅱ核苷酸序列之比較….68 表七 分析親源關係所取樣之菌種及其代號………………………….69 圖目錄 圖一 新日本靈芝菌絲體純培養性狀………………………………….50 圖二 環紋靈芝菌絲體純培養性狀……………………………………51 圖三 新日本靈芝的扣子體……………………………………………52 圖四 新日本靈芝的腫大細胞…………………………………………52 圖五 環紋靈芝的扣子體………………………………………………53 圖六 環紋靈芝的腫大細胞……………………………………………53 圖七 新日本靈芝菌絲體溫度生長試驗結果(培養於MEA12天)……55 圖八 新日本靈芝菌絲體溫度生長試驗結果(培養於PDA12天)……55 圖九 環紋靈芝菌絲體溫度生長試驗結果(培養於MEA12天)………56 圖十 環紋靈芝菌絲體溫度生長試驗結果(培養於PDA12天)………56 圖十一 溫度對新日本靈芝菌絲生長速率之影響……………………57 圖十二 溫度對環紋靈芝菌絲生長速率之影響………………………58 圖十三 酸鹼度對新日本靈芝及環紋靈芝菌絲生長速率之影響……59 圖十四 葡萄糖濃度對新日本靈芝及環紋靈芝菌絲生長速率之影響.60 圖十五 不同碳源對新日本靈芝及環紋靈芝菌絲生長速率之影響…61 圖十六 無機氮源濃度對新日本靈芝及環紋靈芝菌絲生長速率之影響......62 圖十七 酸鹼度對新日本靈芝及環紋靈芝菌絲生長速率之影響…..63 圖十八 PCR產物水平式洋菜膠電泳結果………………………….64 圖十九 分析親源關係所取樣之靈芝屬菌種rDNA之ITS I序列比對…70 圖二十 ITS I序列親源關係圖…………………………………………72

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