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研究生:石辰唯
研究生(外文):Chen-Wei Shih
論文名稱:斜紋夜蛾核多角體病毒紫外線保護劑與量產之研究
論文名稱(外文):Studies on the UV protectant and mass production of Spodoptera litura nucleopolyhedro virus
指導教授:黃榮南
指導教授(外文):Rong-Nan Huang
口試委員:許如君段淑人李松泰石正人
口試日期:2013-06-27
學位類別:碩士
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:2013
畢業學年度:101
語文別:中文
論文頁數:68
中文關鍵詞:斜紋夜蛾核多角體病毒包涵體青春激素活性殘存率紫外線保護劑二氧化矽
外文關鍵詞:Spodoptera lituranucleopolyhedro virusJuvenile hormoneoriginal activity remainingUV-protectantsilicon dioxide
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由於特有的專一性與致病力,且對環境無污染,使桿狀病毒成為一種深具潛力的生物殺蟲劑。然而桿狀病毒實際應用於田間蟲害防治卻不普遍,主因不易大量生產桿狀病毒;此外,病毒對紫外線的抵抗能力普遍不佳,受到紫外線照射後容易喪失活性;目前最符合經濟效益的桿狀病毒量產方法仍倚賴活體幼蟲進行生產,然利用昆蟲幼蟲當作生物反應器來培養包涵體 (occlusion bodies, OBs), 必須要先飼育大量健康的幼蟲,再於感染桿狀病毒後,蒐集垂死或死亡之幼蟲進行病毒純化。過去的研究主要著重於找尋適合的飼養容器、測試最適當的接種量或是其他處理過程的嘗試,包括改變飼養的溫度或是收取病毒的時間點。本論文主要著重於最佳化培養條件和操縱其內分泌系統,來提升斜紋夜蛾重要病原體-核多角體病毒 (Spodoptera litura nucleopolyhedrovirus, SpltNPV) 的產量。由於幼蟲死亡前的體重 (個體大小) 與可獲得的病毒數量呈現正相關性,因此我們利用青春激素 (juvenile hormone, JH) 與青春激素類似物 (juvenile hormone analogs, JHA)-百利普芬 (pyriproxyfen) 來增加幼蟲生長齡期、延遲化蛹的時間,結果顯示最後幼蟲體重與每隻幼蟲獲得之病毒包涵體重量增加2.26與1.95倍,病毒包涵體數量增加2.23與1.97倍。此外,本論文也利用二氧化矽包覆病毒後再加入綠茶,使綠茶吸附於包埋病毒之二氧化矽上,結果顯示二氧化矽包覆病毒以及二氧化矽包覆病毒後吸附綠茶,於照射紫外光 (24000 μJ/min, 254 nm) 90分鐘後之活性殘存率 (original activity remaining, %OAR) 仍有40 與 72%,於紫外線照射180分鐘後OAR為 32 及 60%。市面上常用之紫外線保護劑二氧化鈦與木質素包埋之病毒在經紫外線照射90與180分鐘後,其活性殘存率分別為54 及 55%,以及40 與 36%,由此可知二氧化矽包埋病毒後,再吸附綠茶,可以有效提升對紫外線之保護能力。

Baculovirus have long been recognized as an environmental friendly bio-insecticide, because they infect invertebrate only. The pathogenicity and specificity of baculoviruses have led to them being the alternative to chemical synthetic insecticides which always develop widespread resistance quickly. However, the practical use of baculovirus in pest control has been rather limited due to the difficulty of mass production and sensitive to ultraviolet (UV) radiation of virus. In vivo propagation in living insect is still the most reliable and economic strategy for mass production of baculovirus. Using living insects as a bioreactor for baculovirus production requires a large insect colony as a source of healthy larvae for virus occlusion bodies (OBs) inoculation. Afterward, the infected organisms are harvested as moribund or dead infected insects for virus extraction. The Spodoptera litura (F.) is a polyphagous, multivoltine and worldwide agricultural pest. Therefore, my studies focus on increasing the production of S. litura nucleopolyhedro virus (SpltNPV) by using Juvenile hormone (JH) and its analogs (JHA)-pyriproxyfen. In addition, my thesis also aims to explore silicon dioxide (SiO2) as a UV-protectant for SpltNPV. The preliminary results showed that JH- and pyriproxyfen-treatment can increase the virus production 2.26-folds and 1.95-folds (by weight), and 2.23-folds and 1.97-folds (OBs/larva) respectively. Moreover, the original activity remaining (%OAR) after 90 and 180 min UV radiation can be 40 and 32% respectively when the SpltNPV were encapulated with SiO2. The original activity remaining (%OAR) can be further enhanced to 72 and 60% respectively when the SpltNPV were further treated with green tea (1%) for 30 min. Instead of green tea, titanium dioxide and lignin treatment can only increase the %OAR to 54 and 55%, and 40 and 36% after radiation with UV for 90 and 180 minutes respectively.

誌謝..................................................I
摘要.................................................II
Abstract...........................................III
目錄.................................................IV
壹、緒言.................................................1
貳、往昔研究.............................................6
一、斜紋夜蛾之防治......................................6
二、太陽光與紫外線對生物殺蟲劑之影響........................7
三、紫外線保護劑........................................7
四、二氧化矽...........................................8
五、核多角體病毒的大量生產................................9
六、青春激素與青春激素類似物..............................9
參、材料與方法...........................................10
一、試驗材料...........................................10
二、紫外線保護劑生物活性試驗方法...........................14
三、增加斜紋夜蛾核多角體病毒產量試驗方法....................15
四、斜紋夜蛾核多角體病毒對幼蟲後續發育之影響.................17
五、斜紋夜蛾核多角體病毒分離與純化.........................17
六、資料處理與分析......................................18
肆、結果................................................19
一、二氧化矽與綠茶提升斜紋夜蛾抗紫外線能力試驗...............19
二、增加斜紋夜蛾核多角體病毒產量試驗.......................33
三、斜紋夜蛾核多角體病毒感染對幼蟲後續發育之影響.............43
伍、討論................................................45
一、新型紫外線保護劑的開發...............................45
二、利用百利普芬大量生產病毒..............................46
三、核多角體病毒的垂直感染效益............................48
四、核多角體病毒的儲架期.................................49
陸、引用文獻.............................................51
柒、附錄................................................57


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