2008-11-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/711423摘要：植物交互保護是藉由感染一種與強系病毒親緣相似的弱系病毒所產生的植物抗性。雖然植物交互保護的現象已被發現許久，但是其機制仍然尚未清楚。我們將研發蕪菁嵌紋病毒 (Turnip mosaic virus; TuMV) 的弱系病毒 (mild strain)，利用定點突變的方法，針對 TuMV 的 HC-Pro 三個高度保守的胺基酸 (R180, F205, E396) 進行不同組合的突變。所得的不同突變病毒將測試其在擬南芥植物 (Arabidopsis) 上的致病性 (pathogenicity) 與病毒力價 (titer) 分析，並從這些人工突變的病毒中篩選合適的 TuMV 弱系病毒做為本研究的模式病毒 (model virus)。 本研究也將使用不同波長的螢光基因來標識弱系與強系病毒，以利在共同感染的細胞中區別兩者病毒的分佈。本研究將證明 potyvirus 所誘導之植物交互保護機制是否以基因靜默誘導 (gene silencing) 和蛋白誘導 (protein-mediated resistance) 的雙重抗病機制。在基因靜默誘導的交互保護方面，將使用基因靜默之擬南芥突變株來進行實驗，以驗證參與基因靜默機制的基因是否也參與交互保護抗性。另外，表現 TuMV 及矮南瓜黃化嵌紋病毒 (Zucchini yellow mosaic virus; ZYMV) 之病毒蛋白的轉基因植物，例如突變之 HC-Pro，也將用來驗證蛋白誘導的抗性是否也參與植物交互保護抗病機制。本研究是第一個以 TuMV 弱系病毒與擬南芥突變株建立交互保護的研究平台。預期可以找出參與植物交互保護的基因。此外，本研究所開發的人工突變的弱系病毒亦可應用於十字花科作物上進行交互保護，以作為抗病毒之策略。<br> Abstract: Cross-protection refers to the protection of a plant from virus infection by prior infection with an attenuated strain of the same virus. Although the phenomenon of cross-protection has been known for sometime, little is known about its mechanism of action. We will develop several Turnip mosaic virus (TuMV) mild strains using site-direct mutagenesis on three highly conserved amino acids (R180, F205, and E396) of HC-Pro in which is a viral gene-silencing suppressor of potyvirus. These mutant viruses will be selected as a mild strain by valuation the degrees of pathogenicity and virus titer on Arabidopsis plants. We will attempt to clarify whether the mechanism of cross-protection is mediated by RNAs or proteins or both. The use of different florescence reporters to tag mild and severe strain of viruses will allow us to monitor the distribution of the two virus strains in co-infected tissues. In addition, we will use Arabidopsis mutants affected in specific of siRNA and miRNA pathways to identify gene needed for cross-protection. Furthermore, the transgenic plants expressing various viral proteins, such as mutated HC-Pro, will be also use to test the protein-medicated resistance in cross-protection. This is the first demonstration that uses the mild strain of TuMV and Arabidopsis to study cross-protection. We believe some candidate genes that involved in cross-protection will be identified in this research. In addition, the mild strain of TuMV can also be used for cross-protection in crucifer crops.交互保護基因靜默miRNAsiRNAcross-protectiongene silencingmiRNAsiRNA