臺灣博碩士論文加值系統

以組織切片與類酵母菌共生物 (yeast-like symbiote, YLS) 尿酸酶之基因序列分析，研究稻蝨科 (Delphacidae) 蟲類體內共生物之親緣關係，發現稻蝨科成員體內共生物並不相同，其中11種具有類酵母共生物的種類，進一步利用褐飛蝨 (Nilaparvata lugens) 尿酸酶 (uricase) 基因序列分析其親緣關係。試驗結果顯示稻蝨科YLS之其分類地位屬真菌綱的Ascomycota門，且為單源群，基於Delphacidae內分類群間YLS之相似性，及其YLS之親緣關係系統樹，稻蝨科昆蟲之YLS與寄主昆蟲間具有共同演化之現象，但於蠟蟬總科 (Fulgoroidea) 之各科間，YLS與寄主昆蟲之演化並不一致。同類寄主植物之昆蟲的共生物並無法形成同一單系群，故認為YLS之親緣與昆蟲寄主植物之形成並非同源，可能是長期共同演化下之適應現象。利用in situ hybridization偵測以石蠟包埋之Tarophagus colocasiae及Peregrinus maidis體內尿酸酶基因之位置，結果發現此基因伴隨共生物存在，普遍分布於蟲體各部位含有共生物之體腔內，但在不含YLS之複眼及肌肉等組織則未觀察到，除脂肪體懷菌融合體 (syncytium) 外，在其產卵管、卵巢等組織中累積有較多該基因之DNA。抽取不同齡期褐飛蝨之總體DNA與RNA，再以即時定量PCR分析DNA量及其表現量。結果發現蟲體內的尿酸酶基因DNA量，以雌成蟲及老熟若蟲最多，雄成蟲最少，而體內尿酸酶之mRNA之表現量，則以雌成蟲與初齡若蟲體內最多，老熟若蟲體內最少。將褐飛蝨以35oC 高溫處理1 h之後，再繼續飼育，發現熱處理後在24 h體內mRNA之量最少，但能逐漸回復，至第48及72 h則與對照組相近，此結果顯示熱處理褐飛蝨具有降低共生物尿酸酶基因之功能。本研究結果顯示，YLS尿酸酶基因之DNA量及其表現與齡期有關，且因熱處理會降低其表現，故對於稻蝨科昆蟲具有重要之生理功能。由序列分析之結果亦顯示，YLS與稻蝨科寄主昆蟲間具有某種程度之共同演化關係。

Investigations on taxonomic position and phylogeny of the yeast-like symbiote (YLS) in Delphacidae (Insecta: Hemiptera) were carried out using paraffin sectioning and analyses of uricase gene sequences in the brown planthopper, Nilaparvata lugens. Uricase gene was known to be present in the YLS of the planthoppers (Hemiptera: Delphacidae). Based on uricase gene sequences, the YLS of Delphacidae belongs to Ascomycota in Fungi, and forms a monophylic group. By phylogenetic analysis, coevolutionary relationship between YLS and Delphacidae hosts was suggested, whereas the relationship among the symbiotes of their host insects from the same host plant was placed the same clade. By in situ hybridization, except compound eye and muscle, uricase gene was found in most body cavity, especially abundant in fat body syncytia and also in ovaries and oviposotional tubes. Real-time PCR analyses of the YLS uricase DNA levels and mRNA transcriptions in different developmental stages of N. lugens showed that the uricese DNA was more abundant in adult females than in adult males and the mRNA was declined in the older nymphal stages. After heat treatment at 35oC, the transcription of uricase mRNA was lower in the heat-treated insects than those incubated at room temperature for 24 h. However, the mRNA was restored to the normal level after heat treatment for 48 and 72 h. These results indicated that heat treatment could reduce the activity of YLS uricase gene. The YLS had coevolutionary relationship with Delphicidae hosts based on uricase gene sequence analyses. The DNA and mRNA transcriptions of uricase gene were age dependent, and reduced by heat treatment at 35oC.

誌謝
中文摘要 ……………………………………………… i
英文摘要 …………………………………………… iii
前言 ………………………………………………… 1
文獻摘述……………………………………………… 4
材料與方法 ………………………………………… 17
結果 ………………………………………………… 28
討論 ………………………………………………… 39
結論 ………………………………………………… 52
參考文獻 …………………………………………… 54
圖表 ………………………………………………… 68
附錄 ………………………………………………… 99

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