李後晶臺灣大學:昆蟲學研究所劉耘Liu, YunYunLiu2010-05-102018-06-292010-05-102018-06-292009U0001-2707200915510500http://ntur.lib.ntu.edu.tw//handle/246246/181821在台灣，埃及斑蚊 (Aedes aegypti) 是登革熱病毒的主要傳播病媒。長期以殺蟲劑進行防治工作，在殺蟲劑造成的選汰壓力之下，產生對殺蟲劑具有抗性的族群。 根據世界衛生組織訂定之標準測試程序測試百滅寧對雌成蟲的半數擊昏時間，抗性品系與感性品系兩者在日間對百滅寧的耐受性皆優於夜間，顯示昆蟲對藥劑的耐受性有日週律動。這個現象暗示產生對殺蟲劑具有抗性的抗性基因，可能是受到生物時鐘的調控。以反轉錄聚合酶連鎖反應 (reverse transcription PCR, RT-PCR) 偵測抗藥性基因的表現量，結果顯示在白天CYP9M9的表現量慢慢增加，到了晚上則急速下降，有明顯的日週律動。進一步利用核醣核酸干擾實驗，證明抗性基因CYP9M9的表現具有日夜差異，是受到生物時鐘的控制。以注射period雙股核醣核酸，破壞生物時鐘的運作，觀察CYP9M9的表現。結果顯示注射一天之後，period 的表現量與對照組比較明顯被抑制，CYP9M9的表現量也明顯降低，證明抗藥基因日夜變動是由時鐘基因所調控。本研究探討昆蟲抗藥性與生物時鐘的關係，並從行為、生理與基因層面檢視昆蟲對殺蟲劑抗性的日週律動。The mosquito Aedes aegypti, the major vector of dengue virus in Taiwan, is constantly controlled by insecticides. Under this severe artificial selection pressure, resistant population results in high capability against chemical pesticide. According to the insecticide bioassay, the resistant level of the mosquito against permethrin fluctuated based on the application time of the day. The median knock- down time (KT50) under light condition was significantly longer than that under dark condition in both susceptible and permethrin-resistant strains. This phenomenon implied the expression of the resistant gene underlying the circadian control. The hypothesis would be the expression of permethrin-resistant gene is regulated by clock genes. One of the detoxification genes, cytochrome P450 gene (CYP9M9), expressed in fluctuation within 24 hours which showed high level of expression during day time. To further demonstrate a clock gene, period (per), in controlling the expression of permethrin-resistant gene, RNA interference against per were employed to disrupt the circadian clock. In consequence, the expression of CYP9M9 lost fluctuation and kept at low level whole day. My study provides detail information about circadian control on the expression of a permethrin-resistant gene.口試委員審定書………………………………………..………………………………..i謝…………………………………………………..………………………………….ii文摘要………………………………………..………………………………………iiibstract………………………………………………………………………………….ivontents……………………………………………………………………………….....vist of tables………………………………………………………………………......viiist of figures……….………………………………………………………………..viintroduction……………………………………………………………………………1aterials and Methods ………………………………………………4Mosquito culture…………………………………………………………………....4Total RNA extraction…………………………………………………………….5cDNA synthesis…………………………………………………………………….6Daily fluctuation of gene expression………………………………………………6RNA interference…………………………………………………………………...7Enzyme activity of cytochrome P450………………………………………………9esults …………………………………………………………………………………10stablishment of permethrin-resistant strain…..………………………………….10edian knock-down time (KT50) of permethrin for susceptible and permethrin-resistant strains………………………………......................................11he expression of clock gene period in Ae. aegyptiy…………….……………….13YP9M9 gene expression in mosquito………..……………………………..……16Daily patterns of cytochrome P450 activity in susceptible and resistant strain…..18ffects of period dsRNA injection on the period gene expression………………19ircadian regulation of CYP9M9 gene expression………………………….…….20iscussion………………………………………………………………………………21eferences……………………………………………………………...……………..25ppendix………………………………………….……………………………………32application/pdf540901 bytesapplication/pdfen-US埃及斑蚊百滅寧殺蟲劑抗性日週律動Aedes aegyptipermethrininsecticide-resistancecircadian rhythm[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/181821/1/ntu-98-R96632003-1.pdf