阿拉伯芥中 HC-Pro 介導 RNA 靜默抑制之研究工具

Abstract

目前針對 HC-Pro 介導 RNA 沉默抑制之分子機制研究，仍受限於適當的實驗工具與分析方法。本實驗室先前研究已證實，來自蕪菁嵌紋病毒（turnip mosaic virus, TuMV）的 P1/HC-Pro 蛋白（P1/HC-ProTu）可有效抑制 HEN1 的活性，導致未甲基化 miRNA（unmethylated miRNAs, unMet-miRNAs）累積，並進一步誘導 HESO1 介導的 AGO1 自噬性降解。為建立後續機制研究所需的平台，本研究建立了一套完整的研究系統，包括可作為蛋白質定量內部對照之可靠 ACTIN 抗體、可精確控制 HC-ProTu 時間性表達的誘導型轉基因植物（XVE–P1/HC-ProTu/hen1-8/heso1-1 plant 與 XVE–P1/HC-ProTu/Col-0 plant），以及結合 ContigViews 的轉錄體基因調控網路分析流程，用於解析 RNA 沉默途徑受干擾後所引起的基因表現變化。整體而言，本研究建立了一套整合性的研究平台，可作為未來探討病原如何操控小 RNA 調控途徑，以及 RNA 沉默失調如何重塑植物基因調控網路的重要基礎。

Mechanistic studies of HC-Pro-mediated RNA silencing disruption remain limited by the availability of suitable experimental tools and analytical approaches. Our previous study demonstrated that the P1/HC-Pro protein from turnip mosaic virus (P1/HC-ProTu) is a potent inhibitor of HEN1, leading to the accumulation of unmethylated miRNAs (unMet-miRNAs) and triggering HESO1-mediated autophagic degradation of AGO1. To provide a foundation for future investigations, this study established a research platform consisting of a reliable ACTIN antibody for protein normalization, inducible HC-ProTu transgenic lines including XVE–P1/HC-ProTu/hen1-8-heso1-1, XVE–P1/HC-ProTu/Col-0 plants that allow precise temporal control of HC-ProTu expression, and a transcriptome-based gene network analysis workflow using ContigViews to investigate changes in gene expression associated with perturbation of the RNA silencing pathway. Together, these resources establish an integrated study system that provides a foundation for future studies aimed at understanding how pathogens manipulate small RNA pathways and how RNA silencing disruption reshapes plant gene regulatory networks.