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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 許如君 | |
| dc.contributor.author | Di-Jye Hou | en |
| dc.contributor.author | 侯廸傑 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:17:01Z | - |
| dc.date.available | 2017-08-27 | |
| dc.date.copyright | 2012-08-27 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-18 | |
| dc.identifier.citation | Adams PS. 2007. Data analysis and reporting. pp 39-62. In: Dorak MT (ed). Real-time PCR. Taylor & Francis Press, New York.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63712 | - |
| dc.description.abstract | 東方果實蠅 (oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)) 是台灣重要的果樹害蟲之一，已知為害的寄主植物超過 150 種。合成除蟲菊酯於 2000 年登記為防治東方果實蠅用藥，其作用機制是轉變電壓閥門鈉離子通道 (voltage-gated sodium channel, VGSC) 的構形，並對昆蟲具有擊昏 (knock down) 的作用。Hsu 於室內篩選東方果實蠅抗性品系實驗得知，合成除蟲菊酯產生抗藥性的速度極快，且抗性程度也非常高，因此必須藉著了解東方果實蠅對合成除蟲菊酯的抗性機制，才能有效控管合成除蟲菊酯的抗藥性發生。本論文所使用的東方果實蠅合成除蟲菊酯抗性品系分別為賽滅寧 (cypermethrin (cyp-r))、賽扶寧 (cyfluthrin (cyf-r))、芬化利 (fenvalerate (fenv-r)) 抗性品系以及感性品系。利用局部滴加法測試各個抗性品系對非篩選之合成除蟲菊酯的交互抗性，我們發現所有的抗性品系皆對賽扶寧產生非常高的交互抗性 (cyp-r：62.9 倍；fenv-r：154 倍)，而對賽滅寧與芬化利的交互抗性程度則相對較低。我們以合成除蟲菊酯檢測 cyp-r、cyf-r 與 fenv-r 被擊昏與回復情形，發現cyp-r 與 cyf-r 皆在 30 分鐘內全部昏厥，fenv-r 則約 4 小時才全部昏厥；如事先加上協力劑 PBO 的處理，則會增加抗性品系的被擊昏速度，尤其在 fenv-r 最為明顯；在回復情形部分，發現抗性品系的回復情形略高於感性品系，而 PBO 處理後的所有品系皆無感受性回復之情況產生。利用 PBO、TPP 及 DEM 等協力劑測試對合成除蟲菊酯的協力效果 (synergism)，發現 PBO 不僅在感性品系中有良好的協力效果 (賽滅寧：7.7 倍；賽扶寧：12.1 倍：芬化利：10.4 倍)，且在抗性品系中產生更佳的效果 (cyp-r：26.3 倍；cyf-r：55.6 倍；fenv-r：50.7 倍)。利用不同受質分別測試酯酶 (esterases)、多功能氧化酶、穀胱甘肽硫基轉移酶 (glutathione S-transferases) 的酵素活性，酯酶活性於抗性品系中並沒有顯著提高；利用受質 7-ethoxycoumarin 測試氧化酶活性，cyp-r (3.94 倍) 與 fenv-r (2.31 倍) 顯著高於感性品系，而受質 methoxyresorufrin 則皆無顯著差異。更進一步的利用 Quantitative realtime PCR 去測試 fenv-r 的多功能氧化酶基因，發現 4 個 CYP6 及 1 個 CYP4 家族的氧化酶基因表現量為感性品系的 5 倍以上。在標的作用機制部分，感性品系的除蟲菊酯標的部位電壓閥門鈉離子通道 (Voltage-gated sodium channel, VGSC)已定序完成，序列長度為 6498 個鹼基對，並且比對芬化利抗性品系與感性品系的 VGSC 序列，我們在抗性品系 5 隻個體中發現 R52Q、K437R、V531A、V531T、E1168K、S1449A、S1449T、M1640V、M1640T、V1755I、I1876N 及 D1943G 等胺基酸的變異。由協力劑、酵素活性實驗以及多功能氧化酶基因表現量的分析，我們可以知道多功能氧化酶確實在合成除蟲菊酯抗性品系中，擔任非常重要的角色；標的不敏感抗性方面，雖然我們發現許多只出現在抗性品系中的胺基酸變異，但與目前發表的 kdr 點突變位置並不重疊，而且這些胺基酸變異僅有 I1876N 出現的頻率較高 (4/5)。因此，我們認為室內所篩選出的東方果實蠅合成除蟲菊酯抗性品系的主要機制為多功能氧化酶，而 VGSC 上的胺基酸變異之貢獻仍需更進一步研究。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:17:01Z (GMT). No. of bitstreams: 1 ntu-101-R97632016-1.pdf: 4000903 bytes, checksum: 6d4866788e2041d9ae277e33f671e787 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要… iii 英文摘要 v 本文目錄 ix 表目錄 xii 圖目錄 xiv 本文目錄 1. 前言 1 2. 往昔研究 4 2.1 東方果實蠅的生物學 4 2.2 合成除蟲菊酯 5 2.2.1 合成除蟲菊酯的結構與作用機制 6 2.2.2 合成除蟲菊酯的代謝命運 7 2.3 電壓閥門鈉離子通道 9 2.3.1 電壓閥門鈉離子通道的結構與功能 9 2.3.2 電壓閥門鈉離子的分子特性 10 2.4 合成除蟲菊酯抗性 13 2.4.1 代謝抗性－酯酶 13 2.4.2 代謝抗性－多功能氧化酶 14 2.4.3 代謝抗性－穀胱甘肽硫基轉移酶 15 2.4.4 代謝酵素與協力劑 16 2.4.5 標的不敏感抗性－擊昏抗性 17 2.4.6 果實蠅科對合成除蟲菊酯的抗性研究 18 3. 材料與方法 20 3.1 化學藥劑 20 3.2 東方果實蠅品系 21 3.3 生物檢定 22 3.4 交互抗性測試 23 3.5 協力劑分析 23 3.6 合成除蟲菊酯對合成除蟲菊酯抗性與感性品系的擊昏及其回復反應 24 3.7 代謝酵素活性測試 25 3.7.1 代謝酵素粗萃取 25 3.7.2 酯酶活性測試 25 3.7.3 多功能氧化酶活性測試 27 3.7.4 穀胱甘肽硫基轉移酶活性測試 29 3.8 電壓閥門鈉離子通道序列分析 31 3.8.1 電壓閥門那離子通道的基因資訊與引子設計 .31 3.8.2 Total RNA 萃取 31 3.8.3 反轉錄 PCR 32 3.8.4 電壓閥門鈉離子通道基因定序 33 3.9 合成除蟲菊酯抗性與感性品系之電壓閥門鈉離子通道基因比較 35 3.10 多功能氧化酶基因表現量比較 36 4. 結果 39 4.1 合成除蟲菊酯抗性品系之抗性程度 39 4.2 合成除蟲菊酯間的交互抗性 39 4.3合成除蟲菊酯對合成除蟲菊酯抗性與感性品系的擊昏及回復反應 40 4.4 協力劑測試 40 4.5 酯酶蛋白質濃度與活性測試 41 4.6 多功能氧化酶蛋白質濃度與活性測試 41 4.7 穀胱甘肽硫基轉移酶蛋白質濃度與活性測試 42 4.8 芬化利抗性品系多功能氧化酶基因表現量 42 4.9 Voltage-gated sodium channel 序列分析 43 4.10合成除蟲菊酯抗性品系與感性品系voltage-gated sodium channel 序列比較 43 5. 討論 45 參考文獻 79 附錄 96 表目錄 Table 1. Resistance ratios of three pyrethroid-resistant lines to their own selected pyrethroids 56 Table 2. Cross resistance among cypermethrin-resistant strain, cyfluthrin-resistant strain, and fenvalerate-resistant strain in three pyrethroid insecticides 57 Table 3. Synergism effect to three pyrethroids (cypermethrin, cyfluthrin, and fenvalerate) of the susceptible and the selected resistant strains 58 Table 4. Esterases crude protein concentrations and activities of two substrates on the susceptible and three pyrethroid-resistant strains 59 Table 5. Mixed-function oxidases crude protein concentrations and activities of the substrate, 7-ethoxycoumarin of the susceptible and three pyrethroid-resistant strains 60 Table 6. Mixed-function oxidases crude protein concentrations and activities of the substrate, methoxyresorufin of the susceptible and three pyrethroid-resistant strains 61 Table 7. Glutathione S-transferases crude protein concentrations and activities of two substrates of the susceptible and three pyrethroid-resistant strains 62 Table 8. The substitutions of amino acid in five susceptible and fenvalerate resistant strains of B. dorsalis 63 圖目錄 Figure 1. The knock down rate of susceptible, cypermethrin resistant, PBO treated susceptible, and PBO treated cypermethrin resistant strains with LD50 concentration of cypermethrin in 30 minutes 64 Figure 2. The recovery rate of susceptible, cypermethrin resistant, PBO treated to susceptible, and PBO treated to cypermethrin resistant strains with LD50 concentration of cypermethrin in 48 hours. 65 Figure 3. The knock down rate of susceptible, cyfluthrin resistant, PBO treated to susceptible, and PBO treated to cyfluthrin resistant strains with LD50 concentration of cyfluthrin in 30 minutes. 66 Figure 4. The recovery rate of susceptible, cyfluthrin resistant, PBO treated to susceptible, and PBO treated to cyfluthrin resistant strains with LD50 concentration of cyfluthrin in 48 hours. 67 Figure 5. The knock down rate of susceptible, fenvalerate resistant, PBO treated to susceptible, and PBO treated to fenvalerate resistant strains with LD50 concentration of fenvalerate in 30 minutes. 68 Figure 6. The recovery rate of susceptible, fenvalerate resistant, PBO treated to susceptible, and PBO treated to fenvalerate resistant strains with LD50 concentration of fenvalerate in 48 hours. 69 Figure 7. Expression of the fifteen cytochrome P450 genes in the fenvalerate resistant strains compared with the susceptible strain.. 70 Figure 8. Alignment of open reading frame and the translated amino acid sequence of Bactrocera dorsalis voltage-gated sodium channel gene with para gene of Drosophila melangaster 71 Figure 9. Voltage-gated sodium channel in various transcripts of susceptible and fenvalerate resistant strains of B. dorsalis 75 Figure 10. Alignment of four different voltage-gated sodium channel amino acid sequence 76 | |
| dc.language.iso | zh-TW | |
| dc.title | 東方果實蠅對合成除蟲菊酯之抗性機制探討 | zh_TW |
| dc.title | Pyrethroid Resistant Mechanisms of the Oriental Fruit Fly, Bactrocera dorsalis (Diptera: Tephritidae) | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 馮海東,戴淑美,林鶯熹,劉明毅 | |
| dc.subject.keyword | 合成除蟲菊酯抗性,協力劑,多功能氧化酶,電壓閥門鈉離子通道,東方果實蠅, | zh_TW |
| dc.subject.keyword | pyrethroid resistance,synergist,mixed-function oxidases,voltage-gated sodium channel,Bactrocera dorsalis, | en |
| dc.relation.page | 107 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-18 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 昆蟲學研究所 | zh_TW |
| 顯示於系所單位： | 昆蟲學系 | |
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