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標題: | 解析GATA因子參與Notch所涉入之埃及斑蚊生殖調控 Analysis of GATA factor in the Notch-dependent regulation of reproduction in the mosquito Aedes aegypti |
作者: | Li-Chi Hsieh 謝立祺 |
指導教授: | 蕭信宏 |
關鍵字: | 埃及斑蚊,GATA轉錄因子,生殖,黑化,卵殼過氧化酵素, Aedes aegypti,GATA transcription factor,reproduction,melanization,chorion peroxidases, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | Notch signaling pathway是一個高度保留的訊息傳遞路徑,典型的Notch訊息傳遞路徑是藉由配體 (ligand) 與受體 (receptor) Notch相互結合後,引發Notch被切割,再進一步與下游的CSL (CBF1, Suppressor of Hairless, Lag-1) 共同調控下游基因的表現,而近年的文獻也指出Notch可以在無配體結合的情況下,或是不需要CSL的參與,即可誘發下游基因的表現。我們實驗室過去的研究發現,當我們以RNA干擾 (RNA interference, RNAi) 的方式抑制埃及斑蚊 (Aedes aegypti) Notch的表現,會造成部分的蚊卵無法黑化及硬化,且不論其黑化與否,絕大部分蚊卵皆無法順利孵化,同時我們也證實這樣的結果是透過非典型的Notch訊息傳遞路徑 (Notch-JNK signaling) 所調控,但詳細的調控機制至今仍不清楚。為了進一步確認Notch訊息傳遞路徑參與蚊卵黑化及發育的詳細調控機制,我們利用次世代定序 (next generation sequencing, NGS) 來尋找AaNotch訊息傳遞路徑的下游基因。NGS的分析結果發現,當AaNotch的表現被抑制後,埃及斑蚊GATA4轉錄因子也會連帶受到影響,而GATA轉錄因子是一個從酵母菌到脊椎動物中參與許多發育調控的高度保留轉錄因子。因此我們在本研究中將深入探討埃及斑蚊GATA在Notch依賴性的生殖調控中所扮演的角色及其功能性分析。我們發現AaGATA4會進行選擇性剪切 (alternative splicing) 而形成AaGATAa與AaGATAr,其兩者的差別則是AaGATAa比AaGATAr缺少一段N端鋅手指區域 (N-term zinc finger domain)。我們利用即時定量聚合酶連鎖反應分析發現,AaGATAa及AaGATAr在各時期的表現情形有高度相似性,皆在埃及斑蚊吸血後48小時的卵巢內有大量表現,此外,當AaGATAa及AaGATAr的表現分別受到抑制後,都會造成埃及斑蚊產卵量與蚊卵孵化率下降。接著我們進一步觀察蚊卵內AaKLC 2.2與AAEL008779的表現量以釐清GATA對蚊卵內胚胎發育的影響,我們發現AaGATAa及AaGATAr的表現分別受到抑制後亦會影響蚊卵內胚胎的發育以及蚊卵的受精情形,顯示GATA確實會參與在Notch依賴性之生殖調控。另外,我們同時也發現GATA會透過調控卵殼過氧化酵素 (chorion peroxidase, HPX8C) 而非酚氧化酵素 (phenoloxidase, PO) 來影響蚊卵的卵殼黑化。最後我們證實了GATA確實為JNK的下游基因;建立了一套完整的新型非典型Notch訊息傳遞路徑 (Notch-JNK-GATA signaling),有助於我們深入探討埃及斑蚊的生殖調控與卵殼黑化其詳細的作用機制,用以提供未來生物性防治策略發展的方向。 Notch signaling pathway is an evolutionary highly conserved cell-cell signaling pathway. It has been reported that Notch signaling may activate through a ligand or CSL independent (non-canonical) mechanism. Our previous results demonstrated that silencing of Notch in the mosquito Aedes aegypti resulted in significant inhibition of hatching rate (fertility) and egg tanning (melanization). We also demonstrated that this Notch-mediated regulation of mosquito reproduction is governed by the non-canonical Notch signaling pathway (Notch-JNK signaling). However, the detail mechanisms underlying this regulation remain unclear. We made use of the next generation sequencing (NGS) analysis to investigate the downstream targets of Notch signaling pathway in Aedes aegypti. Our NGS analysis revealed that a GATA4 transcription factor was down-regulated in the absence of AaNotch. GATA factors are a ubiquitous family of transcription factors, conserved from yeast to vertebrates, which regulate a variety of developmental processes. In this study, we found that an alternative splicing was made in the AaGATA4, resulted in two isoforms named AaGATAa and AaGATAr. Quantitative Real-Time PCR analysis revealed a highly similarity of expression profile between AaGATAa and AaGATAr. We demonstrated that the transcripts of both AaGATAa and AaGATAr were highly expressed at 48 hours post blood meal in the mosquito ovary. Silencing of AaGATAa and AaGATAr resulted in a significant reduction of fecundity and fertility in the mosquito. Interestingly, silencing of AaGATAa and AaGATAr resulted in the inhibition of embryogenesis and fertilization. These data revealed that the GATA factor participated in the Notch-dependent regulation of reproduction in the mosquito. In addition, we found that GATA factor participated in egg tanning via the regulation of chorion peroxidase rather than phenoloxidase. Also, injection of JNK inhibitor (SP600125) resulted in a down regulation of AaGATAa and AaGATAr. Taken together, we demonstrated that a novel non-canonical Notch signaling pathway (Notch-JNK-GATA signaling) in the mosquito and the data revealed by this study will be crucial for future study on vector competence and vector control in the field. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19060 |
DOI: | 10.6342/NTU201602925 |
全文授權: | 未授權 |
顯示於系所單位: | 微生物學科所 |
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