Commercial Production of Molecular Identification Kit of Quarantine Thrips and Microarray Identified System of Important Thrips

在分子生物學快速發展及傳統分類人才日漸稀少的情形之下，DNA為主的鑑定系統已是未來的主流之一；此一分子鑑定的工作已廣泛的應用在病毒及微生物等領域，在昆蟲的研究上也已累積不少分子資料。本研究以農業上危害嚴重的薊馬為材料，應用分子檢測技術，開發重要性危害薊馬之DNA 特徵資料，針對各重要檢疫薊馬-像西方花薊馬、澳洲疫薊馬、玫瑰花薊馬-開發檢測試劑，應用於進口農作產品之薊馬檢測；並結合廠商現有的晶片技術共同開發多種薊馬DNA分子檢測的技術及商品，除同時檢測多種薊馬外，亦增加此產品的市場需求。我們已在97年度開發出西方花薊馬的檢測試劑，並成功應用於進口農作產品之薊馬檢測，將進一步尋求其市場的開發；此外，我們也已針對8種重要性薊馬的專一性引子序列進行專利的申請。本研究除持續收集建立國內外重要性危害之薊馬資料庫外，將應用已建立的檢疫薊馬 DNA 資料，開發專一性探針及其專利申請。針對重要的檢疫薊馬測試分子檢測試劑的靈敏度試驗，並使用少量及大量的樣本分別進行分子檢測試劑的測試及應用。除各檢疫薊馬檢測試劑之開發外，更進一步建立生物晶片之檢測方法，同時檢測多種重要危害薊馬，與廠商另合作開發晶片檢測商品，並尋求市場。DNA-based identification in organism will be one of the major approaches in the future and has been applied universally in the identificaiton of viral and bacterial species. Molecular identifications in insect were also increasing rapidly. In this study, we develop molecular characters to identify the agricultural important thrips. The identified procedures and kit for quarantine thrips; for example, western flower thrips, Frankilneilla occidental, have been developed and the commercial kit for F. occidentalis was produced for sale. Furthermore, the patent of specific primers of eight important thrips has been applied now. The work herein, DNA sequences of the other important thrips including several quarantine thrips will be continuously established. In the cooperation with enterprise company, the acquired sequences of the quarantine thrips will be used to develop and establish the commercial identification kit. The identified procedures and methods according to small-scale and large-scale thrips sample will be examined, and established in the identification commercial kit. Furthermore, high-throughput identified systems based on microarray using probes of large number of thrips will be developed; and the detective sensitivity of microarry identified system will be furtherly confirmed. Finally, the specific probe in the systematic identified system based on microarray will be tested using a variety of thrips species, then the commercially identified microarry for thrips will be produced after all

Vicariant Effect of Rivers and Ranges in Loxoblemmus Appendicularis Complex: the Possibility of Immigrant , Phylogeography, and the Introgression

本實驗室先前已建立大扁頭蟋蟀種群其特有的種化模式，證明了大扁頭蟋蟀種群因中央山脈及冰河期陸橋等地質事件，分化出東部物種、南部物種及西-北部物種，且此三種之間於實驗室的飼育下可彼此雜交。目前正進行東部及南部大扁頭蟋蟀物種的研究，探討(a)東大扁頭蟋蟀物種的地理親緣及遷移模式；(b)南大扁頭蟋蟀物種的地理親緣及分化模式；(c)確立東大扁頭及南大扁頭蟋蟀物種之新種分類地位；(d)大扁頭蟋蟀種群是否藉由冰河陸橋自大陸進入台灣。本研究計畫基於先前建立之模式及目前之研究結果，進一步探討河域及山脊分割對西-北部大扁頭蟋蟀物種的遷移及分化，並分析各疊域分佈物種間的雜交比例及分佈。第一年將以西-北大扁頭蟋蟀為主，探討(a)西-北大扁頭蟋蟀的地理親緣及遷移路徑；(b)大扁頭蟋蟀遇到寬廣河域時的過河機制；(c)釐清東南亞各國大扁頭蟋蟀與台灣物種的差異性。第二年則以雜交區為主，探討(a)大武地區雜交帶的基因滲入方向及頻度；(b)嘉義-台南-高雄交界雜交帶基因的滲入模式及頻度；(c)西南大扁頭蟋蟀物種的的分佈範圍；(d)西南大扁頭蟋蟀物種之新種分類地位。期望能用二年的時間進一步釐清大扁頭蟋蟀種群地理親緣、遷移及基因滲入等課題，建立直翅類昆蟲在台灣的分化模式

Commercial Production of Molecular Identification Kit of Quarantine Thrips and Microarray Identified System of Important Thrips

DNA-based characteristics have been applied universally in identification of viral and bacterial species and will become one of the major identified approaches in animals. We develop molecular characters to identify the agricultural important thrips. The identified procedures and kit for quarantine thrips such as Frankliniella occidentalis and Thrips imaginis have been developed either for single-species or multiple-species detection in each reaction. Furthermore, patents for specific primers of eight important thrips, for multiplex-PCR of multiple-species identification technique, and that of specific probes for multiple-species in microarray identification technique have been applied, respectively. The work in this year, DNA sequences more than 50 thrips including several quarantine thrips will be continuously established. In assistance of the cooperated company, the established specific sequences of these thrips will be applying in the single-species and multiple-species identification kit and the multiple-species identified microarray. The identified procedures and methods for kits and array based on small-scale and large-scale thrips sample examination will be complete and established. Furthermore, high-throughput identified system based on microarray detection using probes of more than 50 thrips species will be developed and its detective sensitivity will be testified. Finally, the microarray identified system will be tested using a variety of thrips species before the commercially identified microarray is produced.在分子生物學快速發展及傳統分類少人投入的情形之下，DNA為主的鑑定系統已是未來的主流之一；此一分子鑑定的工作已廣泛的應用在病毒及微生物等領域，在昆蟲的研究上也已累積不少分子資料。本研究以農業上危害嚴重的薊馬為材料，應用分子檢測技術，開發重要性危害薊馬之DNA 特徵資料，針對各重要檢疫薊馬-像西方花薊馬、澳洲疫薊馬、玫瑰花薊馬-開發檢測試劑，應用於進口農作產品之薊馬檢測；並結合廠商現有的晶片技術共同開發多種薊馬DNA分子檢測的技術及商品，除同時檢測多種薊馬外，亦增加此產品的市場需求。我們已在98-99年度開發出多種檢疫薊馬單一及複合的薊馬檢測試劑，並應用於進口農作產品之薊馬檢測；也已開發出偵測30物種的薊馬晶片檢測探針；此外，我們也已針對8種重要性薊馬的專一性引子序列及複合檢測試劑及探針進行專利的申請。今年除持續收集建立國內外重要性危害之薊馬資料庫外，將應用已建立的薊馬 DNA 資料，持續各薊馬或複合檢測試劑之開發，進行近50種薊馬的專一性探針研究及其專利申請，並使用少量及大量的樣本分別進行測試及應用，完成生物晶片之檢測方法流程，開發晶片檢測商品，同時檢測多種重要危害薊馬，尋求商業市場

Development of Molecular Identified System for Important Thrips

在分子生物學快速發展及傳統分類人才日漸稀少的情形之下，DNA為主的鑑定系統已是未來的主流之一；此一分子鑑定的工作已廣泛的應用在病毒及微生物等領域，在昆蟲的研究上也已累積不少分子資料。本研究以農業上危害嚴重的薊馬為材料，應用分子檢測技術，開發重要性危害薊馬之DNA 特徵資料，針對重要檢疫薊馬-西方花薊馬-開發檢測試劑，並結合廠商現有的晶片技術共同開發出多種薊馬DNA分子檢測的技術及商品。我們已在96年度開發出西方花薊馬的檢測試劑，並成功應用於進口農作上之薊馬檢測；將進一步尋求其市場的開發。此外，我們也已針對8種重要性薊馬的專一性序列進行專利的申請。今年除持續收集建立國內外重要性危害之薊馬資料庫外，將應用已建立的薊馬 DNA 資料，測試分子檢測系統的靈敏度試驗，使用少量及大量的樣本分別進行分子檢測系統的測試及應用。更進一步建立生物晶片之快速檢測方法，並與廠商合作開發成檢測商品，並尋求市場。DNA-based identification in organism will be one of the major approaches in the futureand has been applied universally in the identificaiton of viral and bacterial species. Inthis study, we develop molecular characters to identify the agricultural important thrips.The identified procedures and kit for quarantine thrips of western flower thrips,Frankilneilla occidentalis, have been developed. Furthermore, the patent of specificprimers of eight important thrips has been applied now. The work in this year, DNAsequences of the other important thrips will be continuously established. Cooperatedwith commercial company, the acquired sequences of thrips will be used to develop andestablish the microarry identified system. The detective sensitivity of microarryidentified system in thrips will be furtherly confirmed. Finally, the systematic identifiedsystem based on microarray will be tested using individual, small-scale, and large-scalethrips sample, then the commercially identified microarry for thrips will be produced

Fulgoromorpha phylogeny on the basis of the mitochondrial DNA sequence analysis

封面 壹、前言 貳、往昔研究 參、材料與方法 一、材料 二、去氧核醣核酸的萃取 三、去氧核醣核酸的複製及純化 四、去氧核醣核酸序列之定序 五、去氧核醣核酸序列分析 肆、研究結果 一、16rDNA序列的分析結果 1、去氧核醣核酸的組成 2、顛換取代(Transversion,TV)及轉換取代(Transversion,TS) 3、去氧核醣核酸於各分類層的變異 4、類緣關係的分析結果 二、COB序列的分析結果 1、去氧核醣核酸的組成及密碼使用情形 2、去氧核醣核酸的變異及其在不同密碼位的演化速率 3、氨基酸的改變 4、類緣關係的分析結果 伍、討論 陸、參考文獻 圖

Cold treatment for disinfecting Oriental fruit fly (Bactrocera dorsalis Hendel) in Honey murcott (Citrus teticulate Blanco C. sinensis Osbeck )

國內係東方果實蠅（Bactrocera dorsalis Hendel）及瓜實蠅（Bactroceracucurbitae Coquillett）發生疫區，國產鮮果外銷常受輸入國限制。茂谷柑由於糖度高風味佳，受消費者喜好。因此，針對具外銷潛力之茂谷柑進行檢疫處理技術之研發，積極爭取解除輸入國檢疫限制，以拓展外銷市場，穩定提高農民收益，打開國際市場。因此針對此水果研發有效之檢疫殺蟲技術。將測試建立果實蠅於茂谷柑內之各齡期蟲之生長發育及時間分佈，以確認後續試驗之卵期及各齡期之時間判定。並以低溫檢疫殺蟲處理之最適齡期供試蟲，進行之東方果實蠅卵期及各齡期幼蟲抗冷性基礎比較試驗。Elimination of Oriental fruit flies ( Bactrocera dorsalis Hendel ) and B.cucurbitae is necessary to evalute the favorite and taste-well honeyMurret (Citrus teticulate Blanco C. sinensis Osbeck) exportation. Thestudy will develop a cold treatment to eliminate these fruit flies andprovide a quarantine treatment in honey Murret which will be benefiton taiwanese farmers. The optimal life stage and development time for thecold treatment on honey Murret will be established, and the tolerancetest will be conducted on each life stages of fruit fly to demonstratethe optimal cold temperature in quarantine treatment. Among egg and threelarval stages, the most tolerant one will be used in the related clodtreatment on honey Murret

外銷荔枝殺蟲處理檢疫技術研發

輸日荔枝經現行蒸熱冷藏複合方式處理後，部份荔枝果皮產生褐化現象，常影響輸出品質。是以，提列改善荔枝檢疫殺蟲處理計畫，尋求另一檢疫殺蟲處理模式，以解決現階段輸日荔枝經檢疫處理後品質不佳之問題。本試驗即針對4種包括黑葉、桂味、糯米及晚種等不同品系荔枝研發有效之檢疫殺蟲技術。今年檢疫殺蟲處理之小規模、大規模殺蟲試驗並進行果實品質障害試驗。The litchi browniztion after vaporized treatment and cold conservation will influence their export quality. The study will design the methods to promote the quality of four races of litchi after quarantine treatment. The primary examine will be focused on the small- and large-scale treatment of each infetected Bactrocera dorsalis Hendel stages. The fruit quality will be also evalution