2009-08-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/663227摘要：在植物與病毒攻防之間，植物本身具有的防禦反應可抵抗病毒入侵。然而，病毒本身也有反抗能力，可抵抗植物的防禦反應。其最為人熟知的攻防策略就是植物的後轉錄基因靜默機制 (post-transcriptional gene silencing; PTGS) 受到病毒基因靜默抑制子 (viral gene silencing suppressor) 所抑制。Potyvirus 的協同性蛋白水解&#37238; (help-component proteinase; HC-Pro) 是第一個被證明的病毒基因靜默抑制子可抑制植物的 PTGS pathway。PTGS pathway 可大致區分為 microRNA (miRNA) 及 short-interfering RNA (siRNA) pathways。另外，P1 蛋白為 potyvirus 中第一個基因，其緊臨著 HC-Pro，組合成 P1/HC-Pro 的基因體結構。先前研究證實 P1 具有協助 HC-Pro 抑制 PTGS pathway 的能力。P1/HC-Pro 轉基因擬南芥表現鋸齒狀的葉型、葉緣向上捲曲及生長受到抑制等表型，這些不正常的表型也已證實是 P1/HC-Pro 抑制了 miRNA pathway。此外，HC-Pro 突變株 (R180I/F205L/E396N) 顯示失去抑制 miRNA pathway 的能力，但是並沒有失去抑制 siRNA pathway 的能力。根據上述結果，我們推測 P1/HC-Pro 抑制 miRNA pathway 的作用點不同於 siRNA pathway。因此，我們假設有不同 miRNA 與 siRNA pathway 的元件蛋白 (component) 與 P1/HC-Pro 相互作用而導致 PTGS 受到抑制。故本研究將利用蛋白質體學、遺傳學、in vivo 與 in vitro pull-down 等方法分離與鑑定 P1 與 HC-Pro 的相互作用蛋白 (interacting protein)。這些相互作用蛋白基因將會進一步探討其參與 PTGS pathway 與植物抗病機制的角色。對於將來研究以PTGS作為抗植物病毒的策略能夠具有更深入的突破與發展。<br> Abstract: Upon virus infection, plants mount a defense response, which can be counteracted by proteins encoded by the offending virus. The best-known example of such an attack and counter attack strategy is the inhibition of post-transcriptional gene silencing (PTGS) by viral suppressor. The help-component proteinase (HC-Pro) is a potyviral suppressor with multiple functions in the virus life cycle. Recent reports have addressed mechanisms surrounding the role of HC-Pro in the suppression of miRNA pathway and also indicated that P1 protein, which is located in N-terminal of HC-Pro enhances the silencing suppression activity. Moreover, several studies also showed that transgenic Arabidopsis plants expressing P1/HC-Pro fusion gene exhibit abnormal leaf-shape, with serrate, up-curling and shrunken leaves. These abnormal phenotypes are typical miRNA pathway defected phenotype which affected by P1/HC-Pro. In addition, the previous results indicated that the triple mutations (R180I/F205L/E396N) of HC-Pro loses the suppression of miRNA pathway but still retains the ability to suppress siRNA pathway. We conclude that P1/HC-Pro silencing suppressor likely has differential sites of inhibitory action in different small RNA pathways. Therefore, we assume that there are different PTGS components in miRNA and siRNA pathways interact with P1 and HC-Pro. In this project we would like to identify these interacting proteins by proteomes approach, genetics study, and in vivo and in vitro pull-down assay. These interaction protein genes will also be studied the function in PTGS pathway and in innate virus defense response.協同性蛋白後轉錄時期基因靜默機制基因靜默抑制子HC-Propost-transcriptional gene sliencinggene silencing suppressor