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研究生:簡鳳吟
研究生(外文):Feng-YinJian
論文名稱:蝴蝶蘭根部共生菌種之多源基因體研究
論文名稱(外文):The metagenomics of mycorrhiza in Phalaenopsis root
指導教授:蔣鎮宇蔣鎮宇引用關係
指導教授(外文):Tzen-Yuh Chiang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:80
中文關鍵詞:大白花蝴蝶蘭多源基因體學分子條碼
外文關鍵詞:Phalaenopsis amabilisMetagenomicsDNA barcodes
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不論蝴蝶蘭之切花或盆花皆具有高度經濟效益,在台灣尤以原生種大白花蝴蝶蘭(Phalaenopsis amabilis)為最大出口品種。自然環境下,因蘭花種子胚乳發育不全,需仰賴與蘭花根部共生的真菌,以利蘭花生長、萌芽和吸收土壤中的養分。為了因應市場大量需求,大白花蝴蝶蘭栽培種的繁殖都是經由無菌組織培養而來,因此相較於野生種可能具有較低的蘭花真菌多樣性(Orchid Mycorrhizal diversity)。然而,尚未有研究探討大白花蝴蝶蘭的野生種或栽培種,其根部蘭花共生真菌之分佈多樣性。因此,本篇研究以栽培種 KC1111 品系做為研究對象,探討是否在栽培種根部仍有蘭菌多樣性。為了確認栽培中根部有蘭菌的存在,我們做了初步測試。首先從根部進行真菌分離培養,共分離出16 種真菌分屬於14屬(菌根菌6 屬,植物致病菌8 屬)。再將蘭根之基因體DNA為模板,分析真菌核醣體轉錄區間(nrITS)。共產生26種分屬於17屬的真菌ITS。之後以四個核基因與兩個粒線體基因為分子條碼,同樣以蘭根之基因體DNA為模板,利用Illumina Genome Analyzer IIx 進行定序,再以 MEGAN分析蘭花根部所含蘭菌多樣性。由於物種鑑定可能受限於真菌分子條碼資料庫之不足,因此計算個別分類群的遺傳距離 ( P-distance ) 來修正實際物種數,合計至少 225 種真菌存在於栽培種根部。出乎意料地,本篇論文發現栽培種蘭根內仍保有多樣真菌共生,且Ceratobasidiu為主要共生菌種,可能有助於協助萌芽、增強根系發育、抵抗非生物性之壓力、並增加養分的吸收和利用。此外,Arthrobotrys與Dactylella兩屬真菌可捕食線蟲,具生物防治之功效;未知功能之Dendroclathra 與Pachytrype也首次發現存在於蘭花根部。以上結果顯示栽培種蘭花仍然對真菌有營養、抗病或生物防治的依賴。再者,五種分子標記各自可產生特定的真菌群,顯示不同的分子標記對於真菌有不同的專一性,所以無法以單一分子標記偵測所有真菌物種。
No matter the cut flowers or potted, Phalaenopsis sp. is the genus has economic value in the international commercial market. Phalaenopsis amabilis is the most output in Taiwan. In nature, Orchidaceae symbiosis with mycorrhizas for growth because mycorrhizas can provides nutrient for germination that Orchidaceae absent. For the requirement of the large market, Phalaenopsis sp. was cultured for breeding by the tissue culture. Compared to the wild species, cultivars may have low diversity of orchid mycorrhizas. Whether the wild Phalaenopsis amabilis or the cultivar, there were not any related researches about the mycorrhizas in the root. Hence, we explore whether the mycorrhizas exist in the KC1111 line. We were pretest for confirming the mycorrhizas existed in the root. There were 16 species belong 14 genus by fungi isolation and 26 species belong 17 genus by sequencing the ITS region. Further, for investigating the metagenomics of mycorrhizas in orchid root by Illumina. Four nucleus genes and two mitochondrial genes were as the DNA barcoding for revealing the mycorrhizal diversity. We also calculated the genetic distance between all sequences in the individual taxa for correct the bias from the dataset. It was estimated at least 230 species exist in the roots of KC1111 line. Overall, this study found the cultivars still retain the variety of mycorrhizas, and the Ceratobasidium is the most fungus in the root. The other hand, Arthrobotrys and Dactylella as the nematode trapping fungi, with the efficacy of biological control and uncultured Dendroclathra and Pachytrype were the first time found in orchid root. In addition, different DNA marker has different specificity, so we cannot detect all fungal species with a single DNA marker.
中文摘要 I
Abstract II
目錄 IV
表目錄 V
圖目錄 VI

壹、 前言 1
一、 蘭花共生菌 1
二、 蘭菌的分類 3
三、 蝴蝶蘭 4
四、 多源基因體學(Metagenomics) 5
五、 DNA條碼 7
六、 研究目的 8
貳、 材料與方法 9
一、 樣本的取得 9
二、 DNA萃取 9
三、 聚合酵素連鎖反應 11
四、 基因Cloning 11
五、 DNA 定序與分析 13
六、 蘭菌的分離 13
七、 多源基因體分析 14
參、 結果 15
一、 蘭花共生菌之分離與初步定序分析 15
二、 多源基因體分析 18
肆、 討論 21
一、 DNA 條碼的限制性 21
二、 共生真菌之社群結構與分佈 23
三、 多源基因體學分析的限制 27
伍、 結論 30
陸、 參考資料 31
柒、 附錄 38

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