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標題: | 埃及斑蚊TAK1及IAP2之功能性分析 Functional analysis of TAK1 and IAP2 in the yellow fever mosquito, Aedes aegypti |
作者: | Hsin-Chin Wang 王信之 |
指導教授: | 蕭信宏(Shin-Hong Shiao) |
關鍵字: | 埃及斑蚊,先天性免疫系統,核醣核酸干擾, Aedes aegypti,Innate immunity,RNA interference, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | Transforming growth factor-β-activated kinase 1 (TAK1)和Inhibitor of Apoptosis Protein 2 (IAP2)曾被證實在果蠅體內會參與先天免疫系統之調節,但是他們在埃及斑蚊(Aedes aegypti)中所扮演的角色卻仍不明。本研究的目地是釐清TAK1 和 IAP2在埃及斑蚊體內參與先天性免疫所扮演的角色。我們首先由埃及斑蚊的基因庫中選殖出TAK1和IAP2的全長,經分析結果發現其蛋白質結構與果蠅的TAK1和IAP2相似。為了確認TAK1以及IAP2是否參與埃及斑蚊IMD pathway之調節,我們首先利用double-stranded RNA (dsRNA)的方式來分別抑制 Aedes aegypti的TAK1和IAP2,之後分別以Escherichia coli (E. coli)以及Staphylococcus aureus (S. aureus)感染蚊子,再計算蚊子的存活率。實驗結果發現埃及斑蚊的TAK1或IAP2被抑制後,感染E. coli比感染S. aureus之存活率有顯著的下降,證明TAK1以及IAP2在埃及斑蚊體內參與IMD pathway。接著我們利用Real-Time PCR來探討在被細菌感染的埃及斑蚊中,TAK1 及 IAP2的表現趨勢。實驗結果顯示TAK1的RNA表現量在感染E. coli後12小時和48小時有兩個表現高峰。而IAP2的RNA表現量在感染E. coli後的24小時會有明顯的表現高峰。同時我們也利用Real-Time PCR分析TAK1 及 IAP2在埃及斑蚊體內不同組織的表現,實驗結果則發現IAP2在感染E. coli後48小時會在中腸(midgut)有大量表現,而TAK1則無論在E. coli或S. aureus感染後,其RNA表現量均無明顯變化。此外,我們同時也探討埃及斑蚊感染細菌後其體內IAP2及TAK1 protein的表現趨勢以及部位。實驗結果顯示在感染E. coli後的24小時開始即有IAP2的表現,其表現量持續增加至感染後72小時。我們也發現埃及斑蚊感染S. aureus後72小時,IAP2只在脂肪體(fatbody)有較多的表現,但感染E. coli後72小時在fatbody及midgut均有較多的表現。而感染E. coli後的24小時即發現TAK1的protein有大量的表現,感染S. aureus則沒有此現象,同時也發現在感染E. coli 72小時後,可以在埃及斑蚊carcass的部分偵測到TAK1 protein的表現。最後,我們也分析IMD pathway的下游基因: Cecropin 1和Defensin 1的表現。我們首先分別以TAK1、IAP2和LacZ的double-stranded RNA進行RNA干擾(RNA interference),三天後以E. coli感染埃及斑蚊。結果發現IAP2及TAK1被抑制後,皆無法使Cecropin 1及Defensin 1的RNA level於被感染後有大量表現的趨勢,反之,控制組的Cecropin 1、Defensin 1 RNA在感染後24小時則有大量表現的現象。綜合本研究結果,我們發現IAP2及TAK1會特別被E. coli所激發,且IAP2的表現會特別集中在埃及斑蚊的脂肪體及中腸。最後,我們證實IAP2及TAK1扮演著調控埃及斑蚊IMD pathway及其下游基因表現的角色。 It has long been recognized that insects harbor a powerful and efficient immune system. The invertebrate immune response is of cellular and humoral nature, but it lacks adaptive features. They make use of two distinct signaling pathways: the Toll pathway and IMD (immunodeficiency) pathway to mount diverse antimicrobial activities. In this study, we identified two components of the IMD pathway, namely inhibitor of apoptosis 2 (IAP2) and transforming growth factor-activated kinase 1 (TAK1) in the yellow fever mosquito Aedes aegypti. The transcripts of both IAP2 and TAK1 were analyzed by Real-Time PCR and were increased after E. coli challenge. Silencing of IAP2 and TAK1 significantly decrease the survival rate upon E.coli, instead of S. aureus, challenge. This suggests that both IAP2 and TAK1 were involved in the expression of IMD-dependent antimicrobial peptides. The translational expression of IAP2 was increased after E. coli challenge over time. Tissue specific expressional analyses were examined and showed that IAP2 was highly expressed in the mosquito fatbody and midgut in terms of transcriptional and translational level. Transcriptional patterns of two antimicrobial peptides, that is Cecropin 1 and Defensin !, were examined. Our results showed that the induction of both Cecropin 1 and Defensin 1 were significantly reduced in IAP2 and TAK1 knockdown mosquitoes after E. coli challenge. Information gathered in this study will pave the way toward the establishment of efficient strategies for vector control by using molecular engineering approaches. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23137 |
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顯示於系所單位: | 微生物學科所 |
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