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研究生:蔡宜君
研究生(外文):Yi-Chun Tsai
論文名稱:台灣黃蝶微孢子(微粒子屬)體外培養及其生命週期與核糖體核糖核酸基因研究
論文名稱(外文):Life Cycle and the Study of Ribosomal RNA Genes in a Microsporidian Isolate (Nosema sp.) from Eurema blanda arsakia by in vitro Cultivation
指導教授:王重雄
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:77
中文關鍵詞:微孢子微粒子細胞核內發育粒線體核糖體核糖核酸親緣關係分析
外文關鍵詞:MicrosporidiumNosemaintra-nuclear developmentmitochondrionribosomal RNAphylogenetic analysis
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本研究成功自台灣黃蝶Eurema blanda arsakia幼蟲體內純化出一種微孢子 (EB isolate) ,並將它以體外培養的方式在黑角舞蛾 (Lymantria xylina) 細胞株 (NTU-LY cell line),及吉普賽舞蛾 (Lymantria dispar) 細胞株 (IPLB-LD 652Y) 中大量繁殖。這種微孢子在形態及 rRNA 基因序列上都顯現微粒子屬 (Nosema) 的特徵。其rRNA基因的排列方式為LSU rRNA-ITS-SSU rRNA-IGS-5S,亦是一個屬於微粒子屬的重要分子特徵。在形態研究上,包括裂殖體 (meront) 、母孢子 (sporont) 、孢子母細胞 (sporoblast) 、及成熟孢子的超微結構,也再次證明此微孢子屬於微粒子屬。針對 rRNA 進行親緣關係比對,發現此微孢子與 N. plutellae, N. spodopterae, 及 N. bombycis 相當接近。此微粒子蟲在吉普賽舞蛾細胞株中繁殖迅速,並且,在感染後約14天,在宿主細胞外會出現營養體期的孢子。這些營養體期孢子為什麼能離開宿主細胞而獨自生存,仍是未知之謎。特別的是,此微孢子在體外繁殖過程中也會進入宿主細胞核。包括孢子母細胞以及母孢子都在細胞核中被觀察到。另外,我們在研究此微孢子生活史的過程中,意外發現它在母孢子的發育過程中,會吞入宿主細胞的組成物,它是為什麼以及如何吞入宿主組成物,也還是一個有趣而未解的疑問。微孢子一直被認為是沒有粒線體的微生物,但是,我們在實驗室中以專門針對粒線體的特殊染劑,對發育中的微孢子進行螢光染色,發現它能被清楚的染上,免疫金的研究也同樣證實黃蝶微孢子體內含有粒線體細胞色素氧化酶 (cytochrome c oxidase) 蛋白。這些粒線體蛋白可能來自宿主被吞入的粒線體。
A microsporidium was isolated from the larvae of the Three Spot Grass Yellow butterfly, Eurema blanda arsakia, and named as an EB isolate. This microsporidian isolate possesses the molecular and morphological characteristics of the genus Nosema. The organization of the rRNA genes is LSU rRNA-ITS-SSU rRNA-IGS-5S, which matches that of true Nosema species. Phylogenetic analysis, based on the sequences of rRNA genes, placed this isolate as closely related to N. plutellae, N. spodopterae, and N. bombycis. Ultrastructures of each development stage of this isolate, including meront, sporont, sporoblast, and mature spore, also confirmed its Nosema character. The in vitro propagation system of this isolate in IPLB-LD652Y (Lymantria dispar) and NTU-LY (Lymantria xylina) cell lines was successfully established. In life cycle study, the development of this microsporidium under an in vitro cultivation system was observed. It was found to develop in the nucleus of the host cell, a circumstance never encountered in other Nosema-genus microsporidia. An extra-cellular vegetative stage of this microsporidium was also found after 14 days of infection. Besides, during our study of their life cycle, we found that both meront and sporont can internalize the host cell component during their development, why and how they internalize those components is still an interesting and unknown question. Microsporidia are long considered as amitocondrate organism, but in our study, the EB isolate can be clearly stained by the mitochondrion specific dye. The results of immuno-gold study also confirm the existence of mitochondrial cytochrome C oxidase in the microsporidia. Those mitochondria protein might come from the internalized host cell components.
壹、前言………………………………………………………………… 1
貳、前人研究…………………………………………………………… 2
2.1 微孢子的特性…………………………………………………………... 2
2.2 微孢子的形態………………………………………………………..… 3
2.3 微孢子的萌發………………………………………………………..… 3
2.4 微孢子的生活史……………………………………………………..… 5
2.5 微孢子的代謝機制…………………………………………………..… 7
2.6 微孢子與粒線體……………………………………………………..… 9
2.7 微孢子體內的粒線質體 (mitosome) ……………………………….… 9
2.8 細胞色素c氧化酶…………………………………………………..… 11
參、材料與方法……………………………………………………..… 12
3.1 罹病台灣黃蝶……………………………………………………..… 12
3.2 昆蟲細胞株……………………………………………………..… 12
3.3 自台灣黃蝶蟲體純化微孢子……………………………………… 12
3.4 體外培養台灣黃蝶微孢子……………………………………… 12
3.5 宿主細胞外發育中微孢子的純化……………………………… 13
3.6 自體外培養系統純化黃蝶微孢子………………………………… 13
3.7 宿主細胞外孢子之核酸萃取……………………………………… 14
3.8 成熟孢子之核酸萃取……………………………………………… 14
3.9 rRNA 基因增幅及定序……………………………………………… 14
3.10 親緣關係分析……………………………………………………… 15
3.11 螢光染色…………………………………………………………… 15
3.12 穿透式電子顯微鏡………………………………………………… 15
3.13 共軛焦顯微鏡……………………………………………………… 16
3.14 免疫金電子顯微鏡………………………………………………..… 16
肆、結果…………………………………………………………………... 18
4.1 細胞外微孢子的確認………………………………………..… 18
4.2 台灣黃蝶微孢子 rRNA 基因…………………………………… 18
4.3 親緣關係分析……………………………………………………… 19
4.4 生命週期與超微結構……………………………………………… 19
4.5 共軛焦顯微攝影…………………………………………………… 21
4.6 免疫金反應………………………………………………………… 21
伍、討論………………………………………………………………… 22
5.1 台灣黃蝶微孢子之生命週期……………………………………….. 22
5.2 台灣黃蝶微孢子之分類地位……………………………………….. 23
5.3 微孢子的代謝與粒線體…………………………………….……..… 24
5.4 微孢子極絲與粒線體……………………………………..………..… 25
陸、參考文獻……………………………………………………………… 26
柒、圖表…………………………………………………………………… 35
附錄一、黃蝶微孢子核糖體RNA基因完整序列……………………………… 73
附錄二、親源關係分析中所使用的LSU rRNA及SSU rRNA基因….… 77
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