| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 94 === The begomoviruses, whitefly-transmitted geminiviruses, are among the most economically important plant viruses worldwide. The purpose of this study is to develop efficient and convenient protein-protein interaction analysis tools for the study of movement functions of monopartite begomoviruses. A fusion protein-expression vector system was constructed by replacing the C-terminus of Cymbidium mosaic virus coat proteins (CyMVCP) with the full-length coat protein of AYVV-PD (GCP). It was found that the fusion protein has a high immunogenic property and produced effective antiserum against the fusion protein, CyCP-GCP, in the rabbit. The expression level of CyCP-GCP in Escherichia coli protein expression system was increased at least two folds, as compared to CP alone, and anti-CyCP-GCP serum has higher specificity and sensitivity for the detection and diagnosis of begomoviruses. In addition, anti-CyMV serum was used in far western blot assays to demonstrate the protein-protein interactions between CyCP-GCP and AV1, the putative movement protein of monopartite begomoviruses. Subsequently, a series of truncation mutants of AV1, CyCP-AV1 N1-53, CyCP-AV1 M25-75, and CyCP-AV1 C54-117, were constructed to differentiate the binding sites. The N-terminus of AV1 (CyCP-AV1 N1-53) was found to interact with CP, although weakly, which is consistent with the model for the cell-to-cell movement of begomoviruses. These findings suggest that the fusion protein detection and analysis system developed in this study provides a promising strategy for the detection of virus infections. In addition, the N-terminal 1-53 amino acid region was fine-mapped to be the domain responsible for the interaction with CP of AYVV. These finding support that AV1 of monopartite begomovirus may be a functional equivalent of movement protein of bipartite begomovirus.
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