請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16032
標題: | Notch參與調控埃及斑蚊胚胎發育之功能性分析 Functional analysis of Notch in the regulation of Aedes aegypti embryogenesis |
作者: | Yu-Ting Liu 劉玉婷 |
指導教授: | 蕭信宏 |
關鍵字: | 埃及斑蚊,Notch,胚胎發育,卵殼黑化, Aedes aegypti,Notch,embryogenesis,chorion melanization, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 蚊子為許多重要疾病傳播的媒介,例如瘧疾、登革熱、日本腦炎、西尼羅熱、黃熱病等。針對瘧疾及登革熱等,目前尚未有可用的疫苗;另一方面,蚊媒及其攜帶的病原對殺蟲劑或藥物產生的抗藥性使得這些疾病每年奪走約兩百萬人的性命。因此,開發新的蚊媒控制策略對於疫情的防治具有迫切的重要性。Notch signaling pathway是演化上高度保留的細胞間訊息傳遞方式,在發育過程中扮演重要的調控角色。在這個訊息傳遞的路徑中,Notch同時扮演了穿膜受體蛋白以及轉錄因子的角色,當其與配體如Delta或Serrate結合後會經過一連串的酶切過程,最後產生的片段---Notch intracellular domain (NICD),會進入細胞核內調控下游基因的轉錄。果蠅的Notch signaling幾乎參與在各組織、器官的發育調控上,包括中樞及周邊神經、翅膀、卵、肌肉等。然而,對於蚊子Notch的相關研究至今仍付之闕如。我們首先觀察埃及斑蚊中Notch (AaNotch)的表現,發現其高度表現於第四齡幼蟲至蛹期階段,而在成蚊階段則主要表現在吸血後24小時的母蚊的卵巢內。免疫螢光染色的實驗結果則顯示出AaNotch主要表現於吸血後follicle中的oocyte內。為了瞭解Notch signaling在埃及斑蚊中扮演的角色,我們利用RNA干擾的方式抑制AaNotch的表現。實驗結果很驚訝地發現,利用RNA干擾的方式抑制Notch的表現後,不僅產卵數量受到抑制,卵的黑化率也顯著的下降,這些沒有黑化的卵亦無法孵化,此外,有黑化的卵其孵化率也顯著下降至10%以下。利用掃描式電子顯微鏡觀察並比較正常的卵及抑制AaNotch後產出的卵,我們發現抑制AaNotch後會導致埃及斑蚊的卵的表面構造受到影響,micropyle不正常的發育可能使精子無法進入與之受精而無胚胎形成。利用50%漂白水脫去卵的外殼後觀察其內部胚胎發育的情形,發現抑制AaNotch後所產出的卵中幾乎沒有正常成熟的胚胎,初步以RT-PCR確認卵內缺少精子(AAEL008779)及胚胎(AaKLC2.2)相關基因的表現,但其確切原因是否為micropyle造成,則有待進一步實驗釐清。本實驗結果除了初步證實Notch在埃及斑蚊中會調控早期胚胎的生成以外,也發現了在果蠅中沒有觀察到的現象,即Notch與卵黑化之間的關係,其中的調控機制本實驗室正積極研究。Notch在埃及斑蚊正常蚊卵的形態及早期胚胎發育的過程中扮演相當重要的角色,這對未來在蚊媒控制策略上提供另一條可行的方向,期許能應用於控制蚊媒疾病的傳播。 Mosquitoes are important vectors for several infectious diseases such as malaria, dengue fever, Japanese encephalitis, West Nile fever, yellow fever…etc. All together kill more than 2 million people a year, due to the unavailability of effective vaccines for malaria and dengue fever and the development of insecticide and drug resistance to vectors and pathogens. Therefore, there is an urgent need to explore any possible avenue for developing novel control strategies against these mosquito-borne diseases. Notch signaling pathway is an evolutionary highly conserved cell-cell signaling pathway, which regulates many events during development. Notch acts as both transmembrane receptor and transcription activator. After binding to its ligand Delta or Serrate, series of cleavages occur and a fragment of Notch, Notch intracellular domain (NICD), translocates to the nucleus for the transcription of downstream genes. In Drosophila, Notch signaling was shown to be participated in the development of many tissues and organs, including central and peripheral nervous system‚ wings, ovary, muscles, etc. However, the roles of Notch in the mosquito remain largely unknown. We first characterize the Notch expression in the Aedes aegpti and showed that Aedes aegypti Notch (AaNotch) was highly expressed at the 4th instar larvae and the pupal stage. Quantitative PCR analysis showed that AaNotch was mainly expressed in ovary 24 hours post a blood meal. Immunofluorescent assay revealed that AaNotch was located in the oocyte of the follicles post a blood meal. To investigate the roles of Notch signaling in the mosquito development, we made use of the RNA interference approach to silence AaNotch. To our surprise, RNAi-mediated silence of AaNotch resulted in the reduction of egg production and the significant reduction of egg tanning. These non-melanized eggs were unable to hatch and the other melanized eggs have significant low hatching rate (<10%). Scanning electron microscopy revealed that the structure of eggs from Notch-silenced mosquitoes was changed. Abnormal development of micropyle may therefore block the entry of sperm and thus the deposited eggs fail to develop into normal embryo. By using 50% bleach to remove the egg shell, we observed the absent of inner material in the eggs from Notch-silenced mosquitoes. On the other hand, we showed that eggs from Notch-silenced mosquitoes were deficient in one sperm (AAEL008779) and one embryo (AaKLC2.2) genes. Our results showed that Notch was participated in the early embryo development in Aedes aegypti and regulated the egg melanization. We have demonstrated, for the first time, the role of Notch in the development of embryo in the mosquito Aedes aegypti. Further study should be addressed to elucidate the mechanisms involved in the regulation of egg melanization by Notch. Data revealed by this study will be crucial for future studies on vectors competence and vector control in the field. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16032 |
全文授權: | 未授權 |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-101-1.pdf 目前未授權公開取用 | 3.04 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。