- 學位論文
Notch與原酚氧化酵素連鎖反應調控埃及斑蚊卵殼黑化作用之探討
Notch-mediated egg tanning is co-operated with prophenoloxidase cascade in the mosquito Aedes aegypti
摘要
蚊子是許多重要傳染疾病的媒介，例如瘧疾、登革熱、黃熱病、西尼羅熱、絲蟲病等。其中只有黃熱病具有效疫苗，而現有的抗瘧疾藥物也開始出現抗藥性的問題，因此，控制病媒蚊的數量是抑制疾病傳播的一個替代性策略。我們希望透過研究埃及斑蚊產卵及發育的相關機制來找出控制病媒蚊族群的可能性。在蚊子的生活史中，蚊卵產出後需要經過一段時間卵殼才會黑化硬化，此時的卵才能夠保護其內的胚胎。有趣的是，我們在先前的實驗中發現，當我們以RNAi的方式抑制埃及斑蚊Notch的表現，會造成部分的卵無法黑化，而且不論這些卵有無黑化，絕大部分的卵皆無法順利孵化。過去的研究證實蚊子體內的黑化作用主要參與先天免疫反應、傷口癒合、表皮硬化以及卵殼黑化，而參與黑化反應的酵素包含原酚氧化酵素(prophenoloxidase, PPO)、多巴脫羧酵素(dopa decarboxylase, DDC)、多巴色素轉換酵素(dopachrome conversion enzyme, DCE)等，但關於黑化反應與卵殼黑化的研究並不多。此外在哺乳類動物及果蠅的研究也發現Notch是一個高度保留的訊息傳遞路徑，在胚胎發育與決定細胞分化扮演重要的角色。因此我們在本研究中想深入探討埃及斑蚊卵殼黑化的調控機制，以及卵殼黑化反應與Notch的關係。我們利用RNAi的方式分別抑制了原酚氧化酵素與多巴脫羧酵素，結果顯示當多巴脫羧酵素被抑制後可見卵殼黑化明顯受到抑制，而不論我們抑制單一或多個原酚氧化酵素，結果卻發現對卵殼黑化無明顯影響，但若我們使用抑制劑來抑制酚氧化酵素，結果顯示卵殼黑化也受抑制；當我們抑制Notch時，結果顯示原酚氧化酵素之RNA表現與酵素活性皆下降，但多巴脫羧酵素之RNA與蛋白質表現並不受影響，故我們認為Notch透過調控原酚氧化酵素的表現來調控蚊卵卵殼黑化。本研究結果將有助於未來應用於病媒傳病能力之研究及病媒控制策略之制訂。
並列摘要
Melanization is a prominent mechanism for insect physiology, such as innate immune response, cuticular sclerotization, wound healing and egg tanning. Egg tanning, which resulted in the hardening of eggshell, is pivotal for the protection of developing embryos from harsh environmental conditions. It has been demonstrated that prophenoloxidase (PPO) and dopa decarboxylase (DDC), both are important components of PPO cascade, are critical for the regulation of melanization. On the other hand, Notch signaling was shown to be a highly conserved signaling pathway and required for embryonic development. Our previous results revealed that silencing of Aedes aegypti Notch by using RNAi-mediated technique resulted in the significant inhibition of egg tanning. Therefore, we attempt to investigate the potential regulatory machinery between Notch-mediated egg tanning and prophenoloxidase cascade. First, we examine the effect of PPOs and DDC in the egg tanning. Our results showed that silencing of DDC resulted in the inhibition of egg tanning in the mosquito Aedes aegypti, whereas silencing of individual PPO or grouped-PPOs had no effect on egg tanning. Injection of a PO inhibitor into female mosquitoes after a blood meal resulted in the inhibition of egg tanning. RT-PCR analysis and PO activity assay both showed that significant decreases in the PPOs expression and the PO activity were exhibited in the absence of Notch. On the contrary, silencing of Notch had no effect on DDC expression by means of RT-PCR and western blot analysis. Therefore these results suggested an important role of PPOs in the Notch-mediated egg tanning. Data revealed by this study will be crucial for future studies on vectors competence and vector control in the field.
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