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dc.contributor.authorYu-Ting Chenen_US
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dc.description.abstract蜜蜂 (Apis mellifera) 體內的細胞色素P450 (cytochrome P450) 在解毒過程中扮演著重要的角色,可用來抵抗農藥和植物毒素;此外,此類基因也參與體內激素,包括青春激素 (juvenile hormone) 和蛻皮激素 (ecdysteroid) 的合成。利用次世代定序 (next generation sequencing, NGS) 分析蜜蜂幼蟲餵食亞致死劑量益達胺後,對初羽化蜜蜂頭部基因變化之資料庫,結果發現許多細胞色素P450的基因在成蟲時期頭部的表達均受到影響,尤其是CYP343A1在藥劑處理後增加了216倍 (Wu et al., 2017)。根據基因BLAST的結果顯示,CYP343A1為一種與昆蟲青春激素合成有關的酵素methyl farnesoate epoxidase。本研究即針對CYP343A1進行探討。首先,我成功地在蜜蜂體內分離出與CYP343A1同源性相當高之全長cDNA,兩者一致性為95%,稱作AmCYP343A2。AmCYP343A2在初羽化蜜蜂頭部內的表達會隨著成蟲日齡的增加而下降。參照先前Wu et al. (2017) 的試驗餵食蜜蜂幼蟲相同劑量的益達胺 (imidacloprid) 並持續四天後,發現成蟲頭部之AmCYP343A2表現量並沒有顯著地增加。然而,測試蜜蜂頭部內四個構造的AmCYP343A2表達量,結果顯示口器和頭殼具有較高的基因表現量,而這兩部分構造都具有外骨骼。根據這結果推測AmCYP343A2表現之變化可能與昆蟲激素之作用有關;因此,接著將與表皮發育有關的激素20-hydroxyecdysone (20E) 處理粉紅眼 (幼蟲封蓋後約六天) 蜂蛹之結果顯示20E (0.1 µg/µL) 可顯著地刺激AmCYP343A2表現量上升,且發育成成蟲的時間較對照組延遲約一天;相反地,處理青春激素類似物 (juvenile hormone analog, JHA) 百利普芬 (pyriproxyfen, PPN),則會顯著地抑制AmCYP343A2的表現量。另外,針對幼蟲封蓋後約四天之白眼蜂蛹進行RNA干擾 (RNA interference, RNAi) 試驗之結果,並未見AmCYP343A2表現量有顯著的差異,且未觀察到外觀上明顯的變異。綜合上述結果而論,AmCYP343A2在昆蟲表皮和口器中具有高的表現量,且受到20E及JHA的影響,到底AmCYP343A2具有何種生理上的意義?目前仍無法下正確結論,僅推測可能在昆蟲表皮的發育過程中發揮一定的影響。zh_TW
dc.description.abstractCytochrome P450s (CYP450s) in honey bee, Apis mellifera, play a crucial role in detoxification to resist various pesticides and phytotoxins; additionally, CYP450s are also involved in the hormone, such as juvenile hormone (JH) and ecdysteroid, biosynthesis. The previous study showed that the expression of many CYP450 genes in the newly emerged adult worker's heads was significantly affected while the bee larvae were exposed to the sublethal doses of imidacloprid, especially CYP343A1 increased by 216-fold after treatment (Wu et al., 2017). According to the BLAST result, CYP343A1 encodes a methyl farnesoate epoxidase, which is involved in insect juvenile hormone synthesis. In this study, CYP343A1 was the subject to be further investigated. Based on CYP343A1, a full-length cDNA was isolated from the honey bee. This isolated cDNA shows 95% identity with the CYP343A1 gene, encoding a methyl farnesoate epoxidase; therefore, I named this gene, AmCYP343A2. Quantitative RT-PCR (qRT-PCR) measurement of the AmCYP343A2 expression in the heads revealed that the gene expression decrease with adult age. To repeat imidacloprid treatment by Wu et al. (2017), 2- to 5-day-old larvae were fed with sublethal doses of imidacloprid (0.5 ng/µL), and surprisingly qRT-PCR measurement showed that the AmCYP343A2 expression was no significant increase. Furthermore, further analysis of expression of AmCYP343A2 in the honey bee head revealed that the mouthparts and cuticle, which both parts contain cuticle, had the highest expression. Based on this result, it is speculated that the expression of AmCYP343A2 may be related to the action of insect hormone. Therefore, the epidermal development-related hormone, 20-hydroxyecdysone (20E), was used to treat the pink-eyed pupa and the result showed that AmCYP343A2 was significantly up-regulated in 20E-treated groups, and the development of adults delayed about one day compared with the control groups. Comparatively, AmCYP343A2 expression could be repressed by the juvenile hormone analog (JHA), pyriproxyfen (PPN). Moreover, the result of the RNA interference (RNAi) tests with dsAmCYP343A2 on the white-eyed pupa showed no significant difference in the expression of AmCYP343A2, and there was no visible change in phenotypes of adult workers as well. Based on the above results, AmCYP343A2 has highly expressed in the honey bee's cuticle and mouthparts and can be influenced by the 20E and JHA, suggesting that AmCYP343A2 might play an essential role in the insect cuticle development. However, what is the physiological function of AmCYP343A2 is not conclusive at the moment, and it can only be speculated that it may play a specific role in the development of insect epidermis.en_US
dc.description.tableofcontents中文摘要 i ABSTRACT iii 圖目錄 vii 表目錄 viii 前  言 1 前人研究 3 蜜蜂之特性及相關研究 3 農藥對蜜蜂影響之相關研究 4 細胞色素P450之相關研究 6 青春激素之相關研究 8 蛻皮固醇 (ecdysteroids, Ec) 之相關研究 10 基因上游調節區 (regulatory region) 與轉錄調節子 (transcription factor) 11 材料與方法 14 一、供試蟲源及室內飼養 14 二、蜜蜂成蟲頭部解剖 14 三、總量RNA之萃取 14 四、RNA定量 15 五、反轉錄作用 (reverse transcription) 15 六、聚合酶鏈鎖反應 (Polymerase chain reaction, PCR) 16 七、DNA電泳 16 八、定量聚合酶鏈鎖反應 (quantitative real-time PCR, qRT-PCR) 17 九、PCR產物回收 17 十、DNA選殖 (cloning)、定序與分析 17 十一、5'端及3'端DNA的快速擴增 (rapid amplification of cDNA ends, RACE) 19 十二、基因上游轉錄因子結合位 (transcription factor binding sites) 分析 20 十三、基因親緣關係樹 (phylogenetic tree) 分析 20 十四、雙股RNA (double strand RNA, dsRNA) 合成 21 十五、益達胺與昆蟲激素之處理 22 十六、數據統計分析 23 結  果 24 一、細胞色素P450 343A2基因之選殖與定序 24 二、AmCYP343A2在蜜蜂體內之表現 25 三、蜜蜂頭部分區之AmCYP343A2基因表現位置 25 四、RNAi干擾對AmCYP343A2基因表現之影響 26 五、幼蟲餵食低劑量益達胺後之AmCYP343A2基因表現情形 26 六、AmCYP343A2基因上游轉錄因子結合位分析 27 七、昆蟲激素對AmCYP343A2基因表現之影響 27 八、處理青春激素類似物對AmCYP343A2基因表現之影響 27 討  論 28 一、AmCYP343A2之選殖與定序 28 二、AmCYP343A2在正常蜜蜂體內與幼蟲接觸益達胺後之基因表現情形 29 三、AmCYP343A2在蜜蜂頭部之表現位置 30 四、AmCYP343A2上游轉錄因子結合位分析 31 五、昆蟲激素對AmCYP343A2表現之影響 32 六、RNAi干擾對AmCYP343A2表現之影響 33 參考文獻 35 圖  表 50 附  錄 68 一、蜜蜂各時期發育及時間對照表 68zh_TW
dc.subjectApis melliferaen_US
dc.subjectcytochrome P450en_US
dc.title蜜蜂細胞色素P450 343A2基因之選殖與表現zh_TW
dc.titleCloning and expression of the cytochrome P450 gene, AmCYP343A2, in the honey bee, Apis melliferaen_US
dc.typethesis and dissertationen_US
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