請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/30460
標題: Application of RAPD to rapid identification of lepidopteran pests - Using three common pests in Taiwan as a model
利用RAPD技術探討數種鱗翅目害蟲快速鑑定之可行性-以三種台灣常見害蟲為模式
作者: Chang, Jer-Cherng
張哲誠
關鍵字: 隨機增幅核酸多態型技術
RAPD
序列特徵化增幅區域
斜紋夜蛾
甜菜夜蛾
玉米穗蛾
SCAR
Spodoptera litura Fabricius
Spodoptera exigua Hubner
Helicoverpa armigera Hubner
出版社: 昆蟲學系
摘要: 為開發利用分生技術快速鑑定鱗翅目 (Lepidoptera) 害蟲,本研究利用隨機增幅核酸多態型技術 (random amplified polymorphic DNA;RAPD) 分析斜紋夜蛾 (Spodoptera litura Fabicius)、甜菜夜蛾 (Spodoptera exigua Hubner) 及玉米穗蛾 (Helicoverpa armigera Hubner),以建立分析方法之基礎。經測試訂定本研究所使用之RAPD條件為每25 μl樣本中含50 ng模板DNA、250 μM dNTPs、1X PCR緩衝液、0.2 μM引子以及1U Taq耐熱聚合酵素,於迴溫反應器中增幅43個循環。在基本狀況確認之後,以80組隨機引子測試斜紋夜蛾、甜菜夜蛾、玉米穗蛾,從RAPD圖譜中加以判定並挑選出適當之引子以為偵測害蟲種類用,從實驗結果得知OPH-3, OPH-4, OPH-5, OPH-7, OPH-17, OPF-1, OPF-3, OPF-4, OPF-5, OPF-12, OPF-13, OPL-1, OPL-7, OPL-9, OPL-11, OPL-15, OPL-20, OPG-1, OPG-4, OPG-5, OPG-6, OPG-10及OPG-11等引子對於三種受測蟲體均具有頗佳之重現性,對於分辨受測三蟲體有極佳之檢測結果。以同母系產下之斜紋夜蛾不同蟲期之蟲體為模板DNA進行RAPD分析,發現並無明顯差異。另以同地區之斜紋夜蛾四母系所產下之個體為模板DNA進行RAPD分析,則發現隨著母系不同,個體間存在些微差異。另以不同地區所採集之斜紋夜蛾個體為模板DNA進行分析試驗,亦發現RAPD圖譜呈現地域性的差異。為解決RAPD較易產生之個體差異問題,故另外進行以斜紋夜蛾等為模式之專一性引子設計試驗,從OPH-1, OPH-5, OPF-6等引子之RAPD圖譜中選取特定之具種代表性之DNA片段,加以回收、選殖與定序後,藉由所得序列設計專一性引子對:SLH-1, SLH-5, SLH-N5以及SLF-6等, 經進行PCR測試後發現,專一性引子在較嚴苛之反應條件下僅會對目標個體放大特定片段,顯示專一性引子之實用性頗高,應可用於特定種之辨別。
In order to develop a rapid method for identification of lepidopteran pests, we tried to use RAPD (random amplified polymorphic DNA) to achieve our goal. The materials of this study were Spodoptera litura Fabicius, Spodoptera exigua Hubner, and Helicoverpa armigera Hubner. The best condition for RAPD-PCR was 25 μl of RAPD reaction mixture contained 50 ng template DNA, 250 μM dNTPs, 1X PCR buffer, 0.2 μM of random primer, and 1 unit Takara rTaq polymerase. RAPD-PCR were performed in PERKIN ELMER 9600 apparatus with 43 cycles. After collected 80 RAPD random primer tests, we identified OPH-3, OPH-4, OPH-5, OPH-7, OPH-17, OPF-1, OPF-3, OPF-4, OPF-5, OPF-12, OPF-13, OPL-1, OPL-7, OPL-9, OPL-11, OPL-15, OPL-20, OPG-1, OPG-4, OPG-5, OPG-6, OPG-10, and OPG-11 which could be used to identify the tested lepedopteran pests. The different stages (egg, larvae, pupa and adult) of S. litura from the same female resulted no distinct differences on their RAPD profiles. However, RAPD results of S. litura that come from 4 different females revealed that there were minimal differences between them. S. litura from different locations in Taiwan showed RAPD polymorphisms. In order to obtain more species-specific primers, we selected dominant RAPD bands and cloned them; after sequencing, we designed several pairs of primers according to the species-specific bands, such as SLH-1, SLH-5, SLH-N5 and SLF-6. After examinations, the results indicate that species-specific primers can specifically amplify the expected DNA fragments under more stringent condition. We firmly believed that the species-specific primers is more suitable for identification of different pests for quarantine application. 英文摘要---------------------------------------------------------------------- 3 前言---------------------------------------------------------------------------- 5 前人研究---------------------------------------------------------------------- 8 材料與方法------------------------------------------------------------------ 13 結果--------------------------------------------------------------------------- 26 討論--------------------------------------------------------------------------- 36 參考文獻--------------------------------------------------------------------- 45 圖------------------------------------------------------------------------------ 52 表------------------------------------------------------------------------------ 78 附錄--------------------------------------------------------------------------- 79
URI: http://hdl.handle.net/11455/30460
顯示於類別:昆蟲學系

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