地錢與阿拉伯芥 RNA 誘導緘默化複合體之功能性研究

Abstract

植物基因靜默系統利用小分子 RNA 和 ARGONAUTE (AGO) 蛋白質，可調節發育過程以及面對生物和非生物逆境時的基因表現。本論文使用專一性的抗體進行 AGO1 免疫沉澱 (immunoprecipitation; IP) 並建立試管外生物活性測試系統，以分析阿拉伯芥和地錢 Marchantia polymorpha 中 RNA 誘導緘默化複合體 (RNA-induced silencing complex; RISC) 的剪切效率。第二章的研究揭示部分馬鈴薯 Y 病毒屬病毒 (Potyvirus) 的基因靜默抑制子 HC-Pro 抑制 AGO1 功能，達到抵抗基因靜默途徑目的。將 HC-Pro 轉基因植物中 AGO1 處理免疫沉澱後分析生化活性，顯示蕪菁嵌紋病毒的 HC-Pro 顯著抑制 RISC 切割，以利病毒入侵寄主植物。論文第三章提出 MpAGO1 在小分子 RNA 結合和 miRNA 剪切中的特性。此外，MIR11707 基因於高溫環境下負向控制 MpAGO1 平衡。結果顯示，地錢在較高環境溫度生長時加速 miR11707*降解，激活 miR11707 負調控 MpAGO1 表現。此外地錢適應溫度升高時雖降低 miRNA 生合成量，依然可穩定與 AGO1 結合。本論文凸顯 HC-Pro 通過抑制 RISC 活性干擾 AGO1 的功能，也同時提 出地錢適應環境溫度升高時，MpAGO1 調節和表型可塑性之間的聯繫。

Plant gene silencing system comprising microRNAs (miRNA) and ARGONAUTE proteins (AGOs) regulates multiple gene in developmental processes, and responses to biotic and abiotic stress. In this thesis, we established an in vitro assay system using specific AGO1 antibodies to analyze the cleavage efficiency of RNA-induced silencing complex (RISC) through AGO1 immunoprecipitation (IP) in different Arabidopsis and Marchantia polymorpha plants. The investigation in Chapter II uncovers a possibility that HC-Pros from different potyviruses evolve distinct suppression mechanisms to invade host plants. The suppression of RISC activity was confirmed through AGO1-IP of three P1/HC-Pro transgenic plants. Our results indicated that only the P1/HC-Pro of turnip mosaic virus harbored a unique ability to repress RISC slicing. In Chapter III, the intrinsic features of MpAGO1 in small RNA sorting and miRNA cleavage were confirmed. The activation of miR11707 through the acceleration of miR11707* turnover negatively regulated MpAGO1 at high ambient temperature. Besides, the reduction of miRNA biogenesis is not correlated to its AGO1 binding efficiency in response to elevated temperature acclimation. Our study highlights a conserved AGO1 regulatory pathway in bryophyte and the homeostasis of MpAGO1 is control by a novel MIR11707 gene. Taken together, this thesis provides the evidence to support HC-Pro mediated AGO1 suppression pathway via the inhibition of RISC activity. The study implies the link between AGO1 regulation and phenotype plasticity in acclimation to high ambient temperature in M. Polymorpha.