摘要:本計畫主要著重於探討 potyvirus 病毒抑制子 (P1/HC-Pro) 在後轉錄時期基因靜默機制 (post-transcriptional gene silencing, PTGS) 研究上所扮演的角色。Processing body (P body) 是在 PTGS 機制中做為 RNA-induced silencing complex (RISC) 的反應位置。本實驗室的初步結果顯示,HC-Pro 與 P body 在植物細胞質中具有細胞空間重疊性。因此 HC-Pro 可能藉由干擾 P body 的運作而抑制 PTGS 作用。本計畫將建立一套系統生物學研究平台,結合高通量定序分析 (high-throughput next generation sequence; NGS)、蛋白質體學及代謝體學等技術,藉由生物資訊分析進行 P1/HC-Pro 與 P body 間的網路連結。另外,P1/HC-Pro 及 P body 的細胞空間重疊分析也將在本研究中完成。高通量轉錄體及小分子核酸資料也將藉由 NGS 及生物資訊平台進行分析。此外,P body、P1 及 HC-Pro 的相互作用蛋白也將利用 LC-MS/MS 技術進行分析。表現 P1/HC-Pro 的轉殖 Arabidopsis 也將以代謝體學平台進行分析,配合前述之基因體與蛋白體資訊,建構彼此相互連結的生物資訊網絡。我們認為透過基因體、蛋白體及代謝體研究可以提供一個更寬廣的系統生物學研究平台,更深入探討 P1/HC-Pro 如何在 P body 中抑制 PTGS。此外,利用病毒 suppressor - p19 結合電晶體場效的奈米晶片技術 (silicon nanowire field-effect transistor; SiNW-FET) 來偵測與 PTGS 相關的小分子 RNA 並且進行病毒 suppressor 的功能性研究。在這個研究中,我們運用了 p19- SiNW-FET 及 NGS 技術發展出一套新的分析技術,應用在非模式植物鑑定 mi-RNA, siRNA 及 tasiRNA 等基因上。其他病毒的 suppressor 同樣也可利用SiNW-FET 技術,研究其針對 RNA 或細胞元件蛋白的作用特性。This proposal is major focus on the suppressor of potyvirus (P1/HC-Pro) that suppress the post-transcriptional gene silencing (PTGS) pathway. The Processing body (P body) is a working place for the PTGS machinery where has the RNA-induced silencing complex (RISC) activity. Our preliminary data showed that the HC-Pro is co-localized with the P body in cytoplasm of the plant cells. It brings a lot of possibility that the HC-Pro might suppress the PTGS by interfering the function of the P body. We are going to setup a serial of systems biology platforms, such as high-throughput next generation sequence (NGS), proteomes, and metabolome approaches to study the related network between the P1/HC-Pro and the P body. In addition, the cellular localization of the P1/HC-Pro and the P body will be done in this study. The high throughput transciptome and small RNA (miRNA and siRNA) profiles will be analyzed by NGS and bioinformatics approaches. Moreover, the P1 and the HC-Pro interacting protein(s), which located in the P body complex, will be identified by LC-MS/MS of proteome. The transgenic Arabidopsis expressing the P1/HC-Pro will be analyzed by metabolome and the profile will be used for construction the communicated network by combined with NGS and proteome profiles. We assume the network between genome, proteome, and metabolome will provide a deep and wild of angle for studying the P1/HC-Pro-mediated PTGS suppression in P body. In addition, we demonstrated the viral suppressor - p19 immobilized on silicon nanowire field-effect transistor (SiNW-FET) and created an ultrasensitive biosensor for detection the PTGS-related small RNA from the total small RNA pool. In this study, we are going to use p19-SiNW-FET and NGS technology to setup a pipeline for identifying the novel PTGS-related small RNA, such as miRNA, siRNA, and tasiRNA etc. in non- or model plants. Moreover, other virus species of suppressor also will be used in the SiNW-FET technology for studying the characterization of their suppression by interacting w
Abstract: This proposal is major focus on the suppressor of potyvirus (P1/HC-Pro) that suppress the post-transcriptional gene silencing (PTGS) pathway. The Processing body (P body) is a working place for the PTGS machinery where has the RNA-induced silencing complex (RISC) activity. Our preliminary data showed that the HC-Pro is co-localized with the P body in cytoplasm of the plant cells. It brings a lot of possibility that the HC-Pro might suppress the PTGS by interfering the function of the P body. We are going to setup a serial of systems biology platforms, such as high-throughput next generation sequence (NGS), proteomes, and metabolome approaches to study the related network between the P1/HC-Pro and the P body. In addition, the cellular localization of the P1/HC-Pro and the P body will be done in this study. The high throughput transciptome and small RNA (miRNA and siRNA) profiles will be analyzed by NGS and bioinformatics approaches. Moreover, the P1 and the HC-Pro interacting protein(s), which located in the P body complex, will be identified by LC-MS/MS of proteome. The transgenic Arabidopsis expressing the P1/HC-Pro will be analyzed by metabolome and the profile will be used for construction the communicated network by combined with NGS and proteome profiles. We assume the network between genome, proteome, and metabolome will provide a deep and wild of angle for studying the P1/HC-Pro-mediated PTGS suppression in P body. In addition, we demonstrated the viral suppressor - p19 immobilized on silicon nanowire field-effect transistor (SiNW-FET) and created an ultrasensitive biosensor for detection the PTGS-related small RNA from the total small RNA pool. In this study, we are going to use p19-SiNW-FET and NGS technology to setup a pipeline for identifying the novel PTGS-related small RNA, such as miRNA, siRNA, and tasiRNA etc. in non- or model plants. Moreover, other virus species of suppressor also will be used in the SiNW-FET technology for studying the characterization of their suppression by interacting with RNA or cellular components.