臺灣博碩士論文加值系統

蘭科植物的種子發育不完全，需依靠與真菌共生幫助種子發芽生長，所有的蘭科植物在生活史中均涉及與蘭菌的共生關係。本研究探討台灣地區原生蘭種根部共生真菌的菌種多樣性及各植株間菌相結構的差異，比較不同蘭種及地域對共生依存關係的影響。分離黃根節蘭及黃鶴頂蘭植株根部內生真菌共26株，以真菌核糖體RNA基因ITS區域序列作為菌種鑑定依據，並輔以UPGMA等系統演化方法以序列分析菌株間親源關係。結果顯示採集自阿里山黃鶴頂蘭與尖石鄉黃根節蘭植株根系菌相以炭角菌為主，其它植株根部菌相則呈現多樣性，較無特定的優勢菌種。菌相依宿主植株種類不同而有所差異，但以炭角菌屬廣泛存在於調查植株根部。另外，自其他18種台灣原生蘭種分離外表型態類似絲核菌的28株真菌菌株，經比對鑑定出被認定為蘭菌的絲核菌類真菌包括Rhizoctonia、Ceratobasidium及Thanatephorus等菌株，另分離出12株在蘭共生菌中研究較少的木黴菌。最後探討真菌核糖體RNA基因ITS 1/ITS 2區域以DGGE系統對菌株分型，可將相同菌種篩選出來，針對蘭科植物共生真菌提供
較快速的初步鑑定方法。

The minute seeds of orchids are nourished by endophytic fungi to germinate during the nonphotosynthetic early stages of development. All orchids studied so far have involved mycorrhizal in its life cycle. The unique characteristics and diversified species of orchid family may attribute to its distinctive relationship with mycorrhizal fungi. In this study, we have investigated the fungal diversity of Taiwan native orchids. Twenty-six isolated fungal endophytes associated with Calanthe sieboldii Decne. ex Regel and Phaius flavus (Blume) Lindl were analyzed for its ITS (internal transcribed spacer) composition for phylogenetic identification. A great variety of endophytic fungi were isolated from orchid roots. The majority of mycobionts were Xylaria and members of the Xylaria were the primary fungi in every roots sampled. Other 28 isolates were obtained from 18 Taiwan native orchid species and isolates were identified as Rhizoctonia, Ceratobasidium and Thanatephorus, which are generally regard as orchid mycorrhiza. The analysis of DGGE using the ITS 1/ITS 2 region showed that DGGE is useful for the rapid screening of orchid entophytes. This study has proposed a rapid way to identify and characterize of orchid associated fungi and potential to bring positive impact on orchid growth and development.

中文摘要…………………………………………………………i
英文摘要…………………………………………………………ii
目錄………………………………………………………………iii
圖目錄……………………………………………………………v
表目錄……………………………………………………………vii
壹、前言…………………………………………………………1
貳、前人研究……………………………………………………2
一、蘭科植物…………………………………………………2
二、研究對象…………………………………………………5
1. 黃根節蘭…………………………………………………5
2. 黃鶴頂蘭…………………………………………………6
三、菌根………………………………………………………6
四、蘭共生菌…………………………………………………9
五、蘭共生菌的分類…………………………………………12
六、蘭共生菌目前研究方向…………………………………14
七、分子鑑定－研究區域及技術……………………………15
1. 限制酶片段長度多型性(RFLP)…………………………20
2. 簡單重複序列(ISSR)……………………………………21
3. 單股構型多型性(SSCP)…………………………………21
八、變性梯度膠體電泳的原理及應用………………………22
參、材料與方法…………………………………………………25
一、研究材料…………………………………………………25
二、實驗藥品…………………………………………………25
三、儀器設備…………………………………………………26
四、實驗方法…………………………………………………27
五、實驗流程設計……………………………………………35
肆、結果與討論…………………………………………………38
ㄧ、真菌菌株ITS rDNA序列比對分析………………………38
二、黃根節蘭與黃鶴頂蘭共生菌相比較……………………44
三、絲核菌菌株篩選…………………………………………53
四、DGGE分型篩選……………………………………………62
1. 選擇分型區域……………………………………………62
2. 決定DGGE水平電泳條件…………………………………63
3. 利用DGGE對未知菌株分型與篩選………………………66
伍、結論…………………………………………………………75
陸、參考文獻……………………………………………………77

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