臺灣博碩士論文加值系統

在台灣南部三個地區所新發現的入侵植物-刺軸含羞木(Mimosa pigra)，以逢機增殖多態性指紋分析[Random amplified polymorphic DNA (RAPD)]可以顯示出西部地區(梓官)和東部地區(達仁和安朔)植株的基因體不同，初步判定台灣南部東西地區的M. pigra入侵是來自入侵不同的事件。從這些外來入侵的M. pigra根瘤中所分離出191株根瘤菌株，以16S rDNA限制性酶切片段長度多態性分析[Amplified rDNA restriction analysis (ARDRA)]、全細菌蛋白質(Whole cell protein)和酵素免疫分析法[Enzyme-linked immunosorbent assay (ELISA)]，這些分離的根瘤菌株全部都是屬於b-根瘤菌群，其中96.3%與Burkholderia sp.較為相似，而3.7%與Cupriavidus taiwanensis較為相似。選用十株代表菌株進行16S rDNA 序列分析，再次確認它們的相似性，結果顯示出Burkholderia根瘤菌株和南美洲含羞草屬根瘤菌的關係非常接近。再利用八株代表性根瘤菌株進行M. pigra的回種試驗，接種Burkholderia菌株比接種C. taiwanensis菌株具有較高的固氮酶活性(乙炔還原反應)。在共生基因方面(nodA和nifH)，證實Burkholderia (PAS44和PTK47菌株)與C. taiwanensis (PAS15菌株)具有結瘤和固氮基因存在，並再利用綠色螢光蛋白質基因 (Green fluorescent protein [GFP]) 標誌與Gus基因(b-glucuronidase)的Burkholderia sp. PAS44菌株接種於M. pigra，在光學、螢光與立體顯微鏡顯示出此菌株是有效的共生結瘤現象。本論文認為在台灣的M. pigra與早期學者研究的含羞草(M. pudica)與美洲含羞草(M. diplotricha)根瘤菌具有不同的共生現象，由此推論台灣新發現的入侵植物M. pigra的Burkholderia根瘤菌株可能亦是源自於南美洲。

Random amplified polymorphic DNA (RAPD) analysis of Mimosa pigra, located in three sites in southern Taiwan, shows that the genome type from western Taiwan (Tzugan) was different from that in eastern Taiwand (Daren and Anso). This suggests separate invasion events. One hundred and ninty one rhizobial isolates from the root nodules of these invasive M. pigra were examined using amplified rDNA restriction analysis (ARDRA), whole cell protein profiles, and enzyme-lnked immunosorbent assays (ELISAs) with specific antibodies against Cupriavidus (syn. Ralstonia or Wautersia) taiwanensis and Burkholderia phymatum. 96.3 % of the isolates were identified as Burkholderia and 3.7 % as C. taiwanensis. The 16S rDNA sequences obtained from some selected strains also confirmed their identities, and showed that the Burkholderia strains were closely related to Mimosa-nodulating strains from South America. Eight of the strains were confirmed to nodulate M. pigra, but the plants inoculated with Burkholderia strains had considerably higher nitrogenase (acetylene reduction) activities than those inoculated with C. taiwanensis. The symbiosis-essential genes, nodA and nifH, were shown to be present in two M. pigra-nodulating Burkholderia strains (PAS44 and PTK47), as well as one C. taiwanensis strain (PAS15). Further confirmation shows that at least one of these strains are genuine symbionts of M. pigra came from inoculation studies with a green fluorescent protein (gfp) transconjugant strain and Gus gene (b-glucuronidase) of Burkholderia strain PAS44, which showed it to be present within symbiosis-effective M. pigra nodules via light, fluorescence and stereo microscopy. This study suggests that the novel invasive plant in Taiwan, M. pigra, has a different “specific symbiont” from the previous, much older invasive plants, M. pudica and M. diplotricha, and that this specific symbiont (or symbionts) is/are most likely to be in the genus Burkholderia. The predominance of Burkholderia in Taiwanese M. pigra suggests that this species has “carried” its symbionts from its native South America rather than entering into associations with the Taiwanese Mimosa symbionts.

目 錄
中文摘要
Abstract
誌謝
目錄
表次
圖次
附錄
壹、 前言
貳、 文獻回顧
一、固氮作用的研究背景
二、根瘤菌的分類
三、豆科植物共生感染之程序
四、根瘤菌共生基因之研究
五、細菌分類之特徵
參、 材料與方法
一、 實驗流程圖
二、 Mimosa pigra根瘤的採集和Mimosa pigra根瘤菌的分離
三、 植物RAPD 基因型分析
四、 土壤基本理化性質測定
五、 根瘤菌菌株特性研究
(一) ELISA Dot blotting
(二) 蛋白質SDS-PAGE電泳圖譜分析
(三) ARDRA (Amplified rDNA Restriction Analysis)
(四) RAPD (Random Amplification of Polymorphism DNA)
(五) 16S rDNA、nodA和nifH gene的定序分析
(六) 定序
(七) 序列資料與演化樹建構
(八) 根瘤菌回種試驗
(九) 利用乙炔還原能力測定不同型根瘤菌之固氮能力
(十) Burkholderia sp. PAS44菌株的抗生素感受試驗
(十一) 自發性突變株篩選(Spontaneous mutant)
(十二) 細菌接合作用(Conjugation)
(十三) 回種到M. pigra
(十四) 觀察根瘤GFP螢光
(十五) Gus-tag根瘤菌接合與轉位作用(Conjugation)
(十六) 根瘤Gus染色

肆、 結果與討論
一、植物型態及群居情形
(一) 植物型態及群居情形
二、土壤基本理化性質測定
三、根瘤菌的分類特性
(一) 根瘤菌株的分離
(二) ELISA Dot blot
(三) 根瘤菌蛋白質電泳圖譜特性分析
(四) ARDRA (Amplified rDNA Restriction Analysis)
(五) 綜合分析結果
(六) 根瘤菌組基因組分型與地緣關係
(七) 16S rDNA的序列分析
(八) nodA gene的序列分析
(九) nifH gene的序列分析
(十) 根瘤菌固氮能力測定
(十一) 將M. pigra採集到菌種接種到M. pudica
(十二) 抗生素感受測定
(十三) 自發性突變菌秼
(十四) 菌株GFP螢光表現觀察
(十五) 根瘤GFP螢光表現觀察
(十六) 根瘤Gus染色表現觀察
伍、 結論
陸、 參考文獻
表 次

表2. 1 自然界中固氮作用之反應途徑及反應速率5
表2.2 1974年時根瘤菌的分類
表2. 3快生型和慢生型根瘤菌之比較
表2. 4 根瘤菌在細菌分類學之Bergey’s Manual的分類位置
表2. 5 2001年後b-Proteobacteria陸續被發表之文獻
表2. 6 已知的各結瘤基因功能或性質
表3. 1 酵母抽出物甘露醇洋菜培養基之成分
表3. 2 SDS-PAGE的成分
表3. 3 植物培養基配方
表3. 4 Gus染色液
表4. 1 在台灣的三個地區所收集土壤，其基本理化性質測定
表4. 2 所有根瘤菌及特徵
表4. 3在三個地點所採集到不同的根瘤菌RAPD型百分比散佈表
表4. 4將Cupriavidus taiwanensis 和 Burkholderia strains 回種到M. pigra
表4. 5 Burkholderia sp. PAS44和Escherichia coli pUT-mini Tn5GFP在不同的抗生素紙綻上，所呈現的不同的抗性
圖 次

圖2. 1 根瘤菌和豆科植物專一性辨識機制的模型
圖2. 2結瘤基因結構
圖3. 1 本研究在台灣三個採樣點
圖4. 1 M.pigra在梓官農田癈棄地生長情形
圖4. 2 M. pigra在安朔河床分佈情形
圖4. 3 M. pigra在達仁山區分佈情形
圖4. 4 不在地區的M. pigra和參考植株(M. pudica)植株的RAPD pattern利用UPGMA平均數關係係數計算出樹狀分型圖
圖4. 5 將M. pigra所採集到的菌株和參考菌株來利用UPGMA的相關係數將蛋白質圖譜計算出樹狀圖
圖4. 6 ARDRA電泳圖
圖4. 7 M. pigra 所採集到的菌株和標準菌之間的ARDRA pattern利用Dice similarity coefficients (SD)來計算平均數關係係數(UPGMA)的類群分佈
圖4. 8 M. pigra所分離191株菌，在台灣的三個採樣點與ARDRA I、II、III、IV四型分配比例關係圖
圖4. 9 RAPD電泳圖
圖4. 10 將所有的M. pigra菌株做RAPD分析
圖4. 11利用Neighbor-joining比較在台灣M. pigra所分離的根瘤菌與相關b-proteobacteria菌屬16S rDNA序列間之相似性
圖4. 12 利用NodA基因將a-和b-根瘤菌做分類演化樹
圖4. 13 NifH 蛋白質序列演化樹
圖4. 14 Cupriavidus strain PAS17與Burkholderia strain PTK1在回種35 天後生長情形
圖4. 15 利用C. taiwanensis和Burkholderia sp.接種到M. pudica，在 5週期間根瘤數目增加情形
圖4. 16 Burkholderia sp. PAS44菌株之gfp標示螢光圖
圖4. 17 螢光根瘤切片圖
圖4. 18 Gus染色根瘤切片圖


附錄
附錄7. 1　四種限制酵素(Alu I 、Cfo I、Msp I、Hinf I)的辨識位置
附錄7. 2 NCBI的寄存編號
附錄7. 3 本研究使用的a 和b-根瘤菌
附錄 7. 4 pUTmini-TnGFP 質體圖
附錄 7. 5 pRG960sd-32 質體圖

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