- 學位論文
Argonaute 抗體生產及 AGO2 在後轉錄時期基因靜默機制功能探討
Generating Argonautes (AGOs) antibodies and investigating AGO2 functions in suppressing post-transcriptional gene silencing
摘要
Argonaute (AGO) 蛋白是構成 RNA 誘導靜默複合體 (RISC) 的核心。 RISC 在植物中的作用之一是通過轉錄後基因靜默 (PTGS) 或轉錄基因靜默 (TGS) 介導抗病毒防禦。但是，某些植物病毒能夠通過表達RNA靜默病毒抑制子 (VSR) 來抑制宿主 PTGS與TGS，促進感染宿主，增加宿主易感性或出現症狀。另外，在一些研究中已經證明了AGO2在抗病毒防禦中的作用，包括對葉片中蕪菁嵌紋病毒 (TuMV) 的抗病毒作用。為了進一步闡明VSRs 對AGO累積的作用，我們製備 α-AGO2、α-AGO3 和 α-AGO10 IgG 抗體。AGOs N 端 (N) 具有高變異性，因此選擇此區域進行重組蛋白表達，以產出高專一性的 IgGs。我們發現 his-AGO2N 和 his-AGO3N 的高甘氨酸區干擾大腸桿菌重組蛋白表達。去除高甘氨酸區後，his-AGO2CoN 和 his-AGO3CoN (CoN； N 端區域的 C 末端) 可順利表達。由於 AGO3 和 AGO10 在阿拉伯芥中的低表達，α-AGO3 和 α-AGO10 沒辦法檢測到內源性蛋白AGO3 和 AGO10。但是，α-AGO2能檢測內源性蛋白 AGO2。Col-0，轉基因的阿拉伯芥株系，表現P1/HC-Pro 基因，是TuMV 的 VSR (P1/HC 植物) ，含有TuMV感染的Col-0被用來檢測AGO2 蛋白累積，我們觀察到P1/HC植物和感染 TuMV 的 Col-0 中 AGO2 蛋白積累減少。但 FPKM (Fragments Per Kilobase of transcript per Million mapped reads) 分析顯示 P1/HC 植物中 AGO2 轉錄子相較於 Col-0 的表現量上升。這些結果表明P1 /HC-Pro可能在轉譯後觸發 AGO2 降解，開啟了在植物與病毒相互作用的新研究方向。之後將進行進一步研究以了解 AGO2 在植物抗病毒反應中的作用。例如，利用受 TuMV 感染時其 AGO1 及 AGO2 小片段RNA (small RNA; sRNA) 圖譜確認及鑑定特定AGO的sRNAs。此外，通過免疫沉澱分析以AGO1或 AGO2為基礎的 RISC差異。
並列摘要
Argonaute (AGO) proteins constitute the core of RNA-induced silencing complex (RISC). One of the roles of the RISC in plants is to mediate antiviral defense by post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS). However, plant viruses are able to inhibit host PTGS and TGS by expressing viral suppressors of RNA-silencing (VSRs) to facilitate their infection, resulting in an increase of host susceptibility or the development of symptoms. To further elucidate the effect of VSRs in AGO accumulation, we generated α-AGO2, α-AGO3, and α-AGO10 immunoglobulins G (IgGs). Notably, we used the N- terminus region (N) of AGOs for recombinant protein expression since it presents high variability, which would eventually allow the generation of IgGs with high specificity. Also, we found that a high-glycine rich region in the N-terminus of AGO2 and AGO3 interfered with his-AGO2N and his-AGO3N expression in E. coli. After the removal of the high-glycine regions, his-AGO2CoN and his-AGO3CoN (CoN; C-terminus end of the N-termini regions) could be expressed. Endogenous AGO3 and AGO10 proteins could not be detected by α-AGO3 and α-AGO10 perhaps due to their low expression levels in Arabidopsis. In contrast, endogenous AGO2 protein could be detected by α-AGO2. Arabidopsis Col-0, a transgenic Arabidopsis line expressing P1/HC-Pro, which is a VSR of turnip mosaic virus (TuMV) (P1/HC plants), and TuMV-infected Col-0 were used to investigate the accumulation of AGO2. We observed a decrease of AGO2 protein accumulation in P1/HC plants and TuMV-infected Col-0. However, the fragments per kilobase of transcript per million mapped reads (FPKM) value of AGO2 transcript showed upregulation in P1/HC plants compared to Col-0. These results suggest that P1/HC-Pro might trigger AGO2 reduction, which has novelty to open a new investigation direction for plant-virus interaction. Further studies such as sRNA profiling of IP AGO1 and AGO2 under TuMV infection will be used to identify and characterize AGO-specific sRNAs as well as AGO1 and AGO2 immunoprecipitation (IP) will be performed to elucidate the differences in AGO1-based and AGO2-based RISCs.