| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Abstract
The replication ability is one of the key determinants for surviving of viruses. The Rep protein, the first expressed by geminiviruses during the early infection stage, is the only virus-encoded protein required for the replication process, including specific cleavage and ligation to initiate and terminate rolling-circle replication, self-interaction, and regulation of the host cell cycles. In this research, the monopartite begomovirus, ageratum yellow vein virus Ping-Tung strain (AYVV-PT) was used to investigate the interactions between Rep proteins and viral DNAs and to develop the prokaryotic replication system based on E.coli/phage M13 to facilitate the study of geminivirus replication. The Rep gene of AYVV-PT was cloned into plasmid pETBlue for expression, and gel-purified Rep proteins were used for producing specific polyclonal antibodies. The Rep gene was further sub-cloned into plasmid pGEX-4T-1 to obtain Rep proteins in native condition by affinity chromatography. The interactions between Rep proteins and viral genomic DNAs were investigated by Nucleoprotein Binding-ELISA (NB-ELISA) and Southwestern blot analyses. Rep protein has the highest affinity to nt. 1522-2731 of AYVV-PT genome under the optimized in vitro binding condition, 20 mM Tris-HCl buffer, pH 7.4 containing 0.4-0.6 % NaCl. The full-length AYVV-PT genome was cloned into phage M13 as a single unit, and specific Digoxygenin-labeled RNA and DNA probes for AYVV-PT were prepared by in vitro transcription and asymmetric PCR. The signals of small DNAs co-migrating with the single-stranded, circular DNAs encpasidated in virus particles were detected by Southern blot analysis, and the accumulation of the small DNAs was revealed by time course analysis. In addition, the small DNAs were further confirmed as single-stranded, circular DNA of AYVV-PT by RNase A, S1 nuclease digestions and PCR followed by nucleotide sequence analysis. The results suggested that the prokaryotic replication machinery of E.coli/phage M13 could support the generation of single-stranded, circular DNA of AYVV-PT in a construct containing only one copy of replication origin and a single unit viral genome. The E.coli/phage M13 system may facilitate the study of geminivirus replication and the development of convenient infection and foreign gene expression systems.
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