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研究生:余俊瑩
研究生(外文):Chun-Ying Yu
論文名稱:東方果實蠅細胞色素 P450 單氧化酶與有機磷殺蟲劑抗藥性之關聯性
論文名稱(外文):Association of cytochrome P450 monooxygenases and organophosphate resistance in Bactrocera dorsalis
指導教授:許如君吳岳隆
指導教授(外文):Ju-Chun HsuYueh-Lung Wu
口試委員:路光暉戴淑美黃榮南
口試委員(外文):Kuang-Hui LuShu‐Mei DaiRong-Nan Huang
口試日期:2018-07-31
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:昆蟲學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:91
中文關鍵詞:次世代定序技術細胞色素 P450 單氧化酶有機磷殺蟲劑桿狀病毒表現系統
DOI:10.6342/NTU201803524
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東方果實蠅 (Bactrocera dorsalis (Hendel) (Diptera: Tephritidae)) 是臺灣最重要的經濟果樹害蟲之一,最常用來防治東方果實蠅的殺蟲劑是有機磷藥劑 (Organophosphates, OPs),然而長時間的使用,致使田間的東方果實蠅對有機類殺蟲劑產生了抗藥性。代謝抗性是最常發生的抗性機制,同時也是抗藥性管理上最大的挑戰。細胞色素 P450 單氧化酶 (cytochrome P450 monooxygenases, CYP) 是生物體內最重要的代謝酵素之一,在殺蟲劑抗藥性上扮演重要角色。利用次世代定序技術 (Next generation sequencing technique, NGS) 及生物資訊技術獲得東方果實蠅有關 CYP 的轉錄子資訊,共有 388 條轉錄子屬於 CYP,並註解成 136 條基因。以 RPKM (reads per kilobase per million mapped reads) 比較有機磷抗性品系與感性品系之間轉錄子的表現量差異,以抗性和感性之間表現量差異 (RPKM ratio) 大於 8 倍作為挑選條件,共挑選出 6 個 CYP 基因,針對這些基因使用即時定量聚合酶連鎖反應 (Quantitative real-time PCR, qPCR) 確認在東方果實蠅抗性品系與感性品系之間的相對表現量,結果顯示有兩個屬於 CYP6 家族 (family) 的基因具有較高表現量差異。在進行功能性分析時,除了選擇 qPCR 具有高表現量基因外,還挑選了擁有最多轉錄子的 cyp6g1。最後 cyp6g1 以昆蟲桿狀病毒表現系統表現出具有活性的重組蛋白。本研究雖然成功表現具有活性的 CYP6G1 重組蛋白,如果要了解是其否代謝有機磷,仍需要再進一步研究。
The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is one of the most economically damaging fruit pests in Taiwan. Organophosphates (OPs) are the main agents used for control of fruit flies. However, long-term use of organophosphates has caused oriental fruit flies to develop insecticide resistance in the field. Metabolic resistance is the most common resistance mechanism and often presents the greatest challenge in management. Cytochrome P450 monooxygenases (CYP) are one of the most important metabolic enzymes in organisms and play a significant role in insecticide resistance. This study used Next Generation Sequencing technique (NGS) and bioinformation technique to obtain transcript data of CYP in oriental fruit flies. There were 388 transcripts belong to CYP and annotated to 136 genes. The study compared RPKM (reads per kilobase per million mapped reads) values of each OP-resistant and susceptible transcriptome and selected transcripts with RPKM ratios (resistant/susceptible) greater than 8 as candidate transcripts for candidate genes; in total, 6 CYP genes were identified, and quantitative real-time PCR was used to confirm the relative expression levels of candidate genes in OP resistance strains compared to susceptible strain. The results of the study showed that two genes belonging to the CYP6 family had higher expression levels in qPCR expression. My function analysis selected the genes with higher expression levels, and also selected the cyp6g1 gene which had the most transcripts. The cyp6g1 was successfully expressed as an active recombinant protein in the baculovirus expression system. Although this study successfully expressed active CYP6G1 recombinant proteins, further research is needed to understand whether it metabolize organophosphate.
目錄
口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
表次 ix
圖次 x
壹、前言 1
貳、往昔研究 3
2.1 東方果實蠅 3
2.1.1 東方果實蠅生物學 3
2.1.2 現有防治方法 4
2.2 有機磷殺蟲劑 4
2.3 昆蟲抗藥性機制 5
2.4 CYP 6
2.4.1 CYP 的由來及命名 6
2.4.2 昆蟲體內的 CYP 7
2.4.3 CYP 之蛋白質表現 7
2.5 次世代定序技術 (Next generation sequencing technique, NGS) 8
2.5.1 定序技術的演進 8
2.5.2 次世代定序技術之特性 9
2.5.3 次世代定序技術與抗藥性基因之研究 9
參、材料與方法 11
3.1 藥品 11
3.2 東方果實蠅品系 11
3.2.1 東方果實蠅飼育方法 11
3.2.2有機磷抗藥性品系篩選 12
3.3 次世代定序結果分析 12
3.3.1東方果實蠅之 CYP 12
3.3.2挑選候選基因 12
3.4 OP 抗藥性相關 CYP 基因驗證 13
3.4.1 反轉錄 OP 抗性品系 13
3.4.2 定量即時聚合酶連鎖反應 (Quantitative Real-time PCR, qPCR) 13
3.5 候選基因定序 14
3.5.1 設計 cyp6a23、 cyp6d5 及 cyp6g1 完整序列引子對 14
3.5.2 聚合酶連鎖反應 (polymerase chain reaction, PCR) 14
3.5.3 接合作用 (ligation) 及轉型 (transformation) 14
3.5.4 質體確認 15
3.5.5 定序結果組裝 15
3.5.6 親緣分析 15
3.6 以 E. coli (BL21 (DE3)) 進行蛋白質表現 15
3.6.1 將 cyp6a23 和 cyp6d5 轉移至表現宿主 16
3.6.2 以 E. coli 表現重組蛋白 16
3.7 重組桿狀病毒之構築 16
3.7.1 建構共轉染轉移載體 16
3.7.2 共轉染 (co-transfection) 17
3.7.3 病毒感染力測試 18
3.8 重組蛋白之表現與純化 18
3.8.1 表現重組蛋白 18
3.8.2 純化重組蛋白 18
3.9 蛋白質電泳 19
3.9.1 SDS-PAGE 蛋白質電泳 19
3.9.2 考馬斯亮藍 R-250 蛋白質染色 (Coomassie Brilliant Blue R-250, CBR) 19
3.10 西方墨點法 (Western blotting) 20
3.11 CYP 活性測定 20
3.11.1 東方果實蠅三氯松抗性及感性品系蛋白質粗萃取 20
3.11.2 蛋白質定量 21
3.11.3 CYP 活性測試 21
肆、結果 23
伍、討論 28
陸、結論 33
柒、參考文獻 73
捌、附錄 85
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