| 摘要: | 由於過去的20 年來，化學農藥的使用已誘導害蟲產生抗藥性，故防治害蟲時所需的化學農藥劑量和施藥頻率都需提高，如此造成化學農藥的過度使用，進而導致環境的嚴重污染。為改善此問題，開發無污染且高效率的生物殺蟲劑來取代化學殺蟲劑，已是當前很重要的研究課題。在過去許多重組桿狀病毒的實驗，均是將多角體基因從桿狀病毒基因組中刪除，再插入外來的抗蟲蛋白或酵素基因，經此重組過程，所產生的重組桿狀病毒將不再產生多角體，故在田間並不穩定。基於此原因，本計畫擬構築含多角體的重組桿狀病毒，已增進其殺蟲效率與田間應用的潛能。在此計畫將討論：1.測試vAcPhsp70EGFP/Ppag90IT2 重組加州苜蓿夜蛾核多角體病毒對其他三種本土夜蛾科昆蟲幼蟲的殺蟲活性測試，包括甜菜夜蛾、斜紋夜蛾、小菜蛾等。2.利用蚜蟲病毒的核醣體內轉譯子建立一含多角體或包含體之重組加州苜蓿夜蛾核多角體病毒 (recombinant polyhedrin-positive AcMNPV)的雙效表現載體系統；使其同時表現二個外源基因，並產生多角體。3.將來自植物愛玉的幾丁質酵素基因插入重組加州苜蓿夜蛾核多角體病毒的雙效表現載體系統，產生vAcP10Chi-IR-EGFP 重組病毒。以測試綠螢光(EGFP)報導基因的表現作為蟲體感染的指標與偵測。4. 利用電子顯微鏡觀察來自愛玉之幾丁質酵素對對擬尺蠖、甜菜夜蛾所造成之病理現象。5.探討polyhedrin 與NPV 其他蛋白之間的關連性與穩定性，因本實驗室執行之重組桿狀病毒均是在含有polyhedrin 的桿狀病毒載體系統，故對polyhedrin 在NPV 的生活史中所扮演之協調與穩定之功能(角色) 的瞭解，將有益於將來在發展含有occlusion bodies 之重組桿狀病毒的研究與實際田間的應用。預期本計畫將可達到下列成果：所建立之雙效表現載體系統，除可同時表現兩個抗蟲或殺蟲基因，產生協同或加成的殺蟲效果外，還可產生多角體或包含體形式的病毒。在田間此含多角體重組病毒較不含多角體重組病毒穩定。重組病毒vAcPhsp70EGFP/Ppag90IT2 和 vAcP10Chi-IR-EGFP 除以綠螢光作為鑑定染病蟲體的指標外，還可增加對農業害蟲的殺蟲效率，縮短害蟲噬食的時間。同時，此經重組的病毒因殺蟲效率高故可減少其使用劑量，相對亦可減少商業生產時所需的經費。因此，開發兼具速效性及監測性的重組病毒將可取代傳統的化學農藥，並落實害蟲綜合管理的理念與宗旨
Many chemical insecticides are no longer effective in the field due to the induced resistance of insects to these compounds abusively used in past decades. In addition, long-term utilization of chemical insecticides has also resulted in serious pollution of the environment. Thereby, searching for new pollution-free and fast-acting insecticides to replace chemical insecticides becomes an important issue for pest control. In past, most experiments of improve bauloviruses activity that the polyhedrin sequence have been deletion from the viral genome for inserted the insect-specific toxin or enzyme, and then to enhance the insecticidal efficacy of baculoviruses. However, the polyhedrin-negative baculoviruses are not stable in the field. For this reason, we will generate the recombinant polyhedrin-positive baculoviruses for keep their stability and enhance their insecticidal efficacy. These topics will be discussed in this study. First, to assay the insecticidal efficacy of recombinant AcMNPV vAcPhsp70EGFP/Ppag90IT2 against Lepidoptera crop pests, including Plutella xylostella, Spodoptera exigua, Spodoptera litura etc. Second, we will employ the internal ribosome entry site (IRES) element of rhopalosiphum padi virus (RhPV) to construct a bi-cistronic expression polyhedrin-positive vector, pAcP10DR-IR-EGFP, for dual foreign protein genes expression and polyhedron production. Third, further to construct a chitinase gene from Ficus awkeotasang Makino into the bi-cistronic polyhedrin-positive recombinant AcMNPV, vAcP10Chi-IR-EGFP, which contain an EGFP as a detect marker for infected insect larva. The fourth, the chitinase from Ficus awkeotasang Makino causes the pathology phenomenon of Trichoplusia ni and Spodoptera exigua larvae was observed by electron microscopy. Finally, we will carry on the study about polyhedrin interaction with other proteins of NPV. It is expected that this study has the following achievements: First, the polyhedrin-positive virus is more stable than polyhedrin-negative virus in the field. In addition, the bi-cistronic expression polyhedrin-positive vector can drive two anti-insect proteins expression, and then producing the coordinative or additional efficacy. Second, the recombinant baculovirus vAcPhsp70EGFP/Ppag90IT2 and vAcP10Chi-IR-EGFP may be significant increase the insecticidal potency and shorten the time of fetch food than wild-type AcMNPV. On the other hand, the green fluorescence can be used for the identification of infected larvae. Third, reduce the dose of use in the field, so it would be reduced the cost of the commercial production. In brief, the quick-acting and detectable of recombinant baculovirus can play an important role in integrated pest management systems and provide a potential to replace the chemical pesticides in pest control. |
