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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 許如君(Ju-Chun Hsu) | |
dc.contributor.author | Ting-Wei Ye | en |
dc.contributor.author | 葉庭維 | zh_TW |
dc.date.accessioned | 2021-06-17T06:00:31Z | - |
dc.date.available | 2022-02-14 | |
dc.date.copyright | 2019-02-14 | |
dc.date.issued | 2019 | |
dc.date.submitted | 2019-02-12 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/71426 | - |
dc.description.abstract | 小菜蛾 (Plutella xylostella) 是現今被認為對十字花科植物最具威脅性的害蟲,對許多不同機制的殺蟲劑都已經產生抗藥性,本論文針對田間常用的四種殺蟲劑 (剋安勃、因滅汀、脫芬瑞、氟大滅) 進行抗性篩選,之後搭配先前實驗室的小菜蛾次世代定序 (Next Generation Sequencing) 資料,分別挑出上述四個殺蟲劑抗性品系與當時第一子代相比 FPKM (Fragments Per Kilobase of transcript per Million mapped reads) 值有 3 倍以上差異,並從每個抗性品系中挑選出 4 個差異表現最高,且屬於細胞色素 P450 單氧化酶 (Cytochrome P450 monooxygenase)、穀胱甘肽-S-轉移酶 (Glutathione S-transferase) 或羧酸酯酶 (Carboxylesterases) 三種常見殺蟲劑代謝酵素類群之基因做為候選基因,結果發現一種羧酸酯酶的表現量在脫芬瑞抗性品系 F5 中有明顯增加,由協力試驗 (Synergism test) 證實羧酸酯酶類群基因被抑制後會影響小菜蛾對脫芬瑞的敏感性,最後再以 dsRNA 干擾此羧酸酯酶基因表現,72 小時後與控制組相比表現量顯著降低了 4.9 倍,並在 RNA 干擾後餵食脫芬瑞,72 小時與控制組相比,干擾此羧酸酯酶表現之小菜蛾於診斷劑量處理死亡率由 4.1% 增加至 43.8%;而以脫芬瑞對此品系 LC50 (Lethal Concentration 50%) 劑量處理死亡率由 46.9% 增加至 85.4%,證實此羧酸酯酶基因確實與小菜蛾對脫芬瑞抗性相關。 | zh_TW |
dc.description.abstract | The diamondback moth (Plutella xylostella) is currently considered the biggest threat to cruciferous plants, as it has developed resistance to many insecticides. This study utilizes insectides (chlorantraniliprole, emamectin benzoate, tolfenpyrad, and flubendiamide) commonly used in the field to select resistant moths. Using Next Generation Sequencing (NGS) data, we compared four resistant strains with our current F1 strain. Four metabolic enzyme genes (cytochrome P450 monooxygenase, glutathione S-transferase, carboxylesterases) with more than 3-fold difference in FPKM values were chosen as candidate gene, and the four insecticides were used for selection. Expression levels of candidate genes in the fifth generation of these strains were tested using quantitative real-time PCR (qPCR) and expression levels of one carboxylesterase was shown to be heightened in tolfenpyrad-resistant strains. Esterase inhibitor TPP (triphenyl phosphate) given during synergism tests was shown to significantly increase tolfenpyrad susceptibility in tolfenpyrad-resistant strains, thereby proving that esterase inhibitors can increase tolfenpyrad susceptibility. Finally, dsRNA interference of carboxylesterase expression showed a 4.9-fold decrease in expression levels after 72 hours in comparison with the control group. The test group was given tolfenpyrad after RNA interference and a post-72h comparison with the control group showed that mortality rates increased from 4.1% to 43.8% under the diagnostic dose and LC50 mortality rates of tolfenpyrad increased from 46.9% to 85.4%, proving that reduction of expression levels in this carboxylesterase also decreased diamondback moth resistance to tolfenpyrad. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:00:31Z (GMT). No. of bitstreams: 1 ntu-108-R03632021-1.pdf: 2084000 bytes, checksum: 94518bcd5602065cf3f98a0208a92ab1 (MD5) Previous issue date: 2019 | 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 iv 目錄 vi 表目錄 viii 圖目錄 ix 第一章 前言 1 第二章 往昔研究 3 2.1 小菜蛾生物學 3 2.2 小菜蛾對殺蟲劑之抗藥性 4 2.2.1 代謝抗性 5 2.2.2 標的抗性 7 2.3 脫芬瑞簡介 8 2.4 抑制粒線體電子傳遞殺蟎劑之抗藥性機制 9 2.5 小菜蛾基因靜默實驗 10 2.5.1 以注射方式沉默小菜蛾基因探討抗藥性機制 11 2.5.2 以餵食方式沉默小菜蛾基因探討抗藥性機制 12 第三章 材料與方法 13 3.1 化學及分生試劑 13 3.2 溪湖品系小菜蛾採集 13 3.3 小菜蛾品系建立與飼養 13 3.4 感受性試驗 14 3.5 脫芬瑞半數致死濃度測定 14 3.6 協力試驗 15 3.7 次世代定序轉錄子的蛋白質功能註解 15 3.8 總 RNA 抽取與轉互補 DNA 15 3.9 脫芬瑞抗性小菜蛾食用半診斷劑量基因表現量差異 16 3.10 聚合酶連鎖反應 16 3.11 定量 PCR 表現量分析 17 3.12 定量 PCR 引子對設計 17 3.13 雙股 RNA 生產 17 3.14 雙股 RNA 餵食溶液製備 18 3.15 RNA 干擾試驗 18 3.16 脫芬瑞半數致死時間測定 18 3.17 79008 基因序列定序與重組殖體建立 19 3.18 親緣關係分析 20 3.19 統計分析 20 第四章 結果 21 4.1 溪湖品系小菜蛾回復品系對四種殺蟲劑之感受性 21 4.2 抗藥性品系篩選 21 4.3 抗藥性品系候選基因相對表現量 21 4.4 79008 於三殺蟲劑抗性品系中 F3、F5 間表現量差異 22 4.5 79008 於脫芬瑞抗性品系中各代間表現量差異 22 4.6 協力試驗 23 4.7 脫芬瑞誘導解毒基因試驗 23 4.8 RNAi 試驗 23 4.9 RNAi 處理後毒性測試 24 4.10 RNAi 處理後對脫芬瑞半數致死時間測定 24 4.11 基因定序、重組與限制酶剪切 24 4.12 脫芬瑞抗性品系 79008 之蛋白質身分鑑定及親源分析 25 第五章 討論 26 參考文獻 54 附錄 74 | |
dc.language.iso | zh-TW | |
dc.title | 一種羧酸酯酶對小菜蛾脫芬瑞抗藥性的影響 | zh_TW |
dc.title | Impact of one Carboxylesterase on Tolfenpyrad Resistance of Plutella xylostella | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 戴淑美(Shu-Mei Dai),楊永裕(Yeng-Yuh Yang),黃榮南(Rong-Nan Huang) | |
dc.subject.keyword | 小菜蛾,殺蟲劑抗藥性,抗性選汰,脫芬瑞, | zh_TW |
dc.subject.keyword | Diamondback moth,Insecticide resistance,Resistance selection,Tolfenpyrad, | en |
dc.relation.page | 78 | |
dc.identifier.doi | 10.6342/NTU201900382 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2019-02-12 | |
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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