| Summary: | 碩士 === 中興大學 === 生物科技學研究所 === 95 === The genus Begomovirus is a member of the family Geminiviridae, consisting of whitefly-transmitted dicotyledonous plant-infecting geminiviruses that are among the most severe plant viruses in the tropics and subtropics. Packaged within geminate particles, the genomes of geminiviruses are single-stranded circular DNAs that are replicated mainly by a rolling circle mechanism, characteristic to prokaryotes. Previous researches have indicated that geminiviruses can generate various DNA replication forms in bacteria, suggesting that geminiviruses possess the potential to regulate the expression of certain viral genes in prokaryotic systems. However, whether additional unknown genes or regulatory sequences are functional in prokaryotes remained to be elucidated. There the aim of this study is to identify and characterize promoter sequences and unknown open reading frames in geminivirus genome in prokaryotic system. Using the whole genome of Ageratum Yellow Vein Virus Ping-Dung Strain (AYVV-PD) as the template for random polymerase chain reaction (PCR), the potential prokaryotic promoter regions were investigated in Escherichia coli. The promoter-trapping vector pGlow-TOPO were employed with green fluorescent protein (GFP) as the reporter. An initial search revealed the prokaryotic promoter sequence between nt 615-889 of AYVV-PD genome, tentatively designated as AV3 promoter, which has no known downstream open reading frame. In order to identify the minimal region of the promoter, the Erase-A-Base system and inverse PCR were used to generate unidirectional deletion mutants with various GFP expression levels reflecting the strength of the promoter. It was revealed that nt 764-837 is the minimal region required for promoter activity. By duplicating AV3 promoter regions in different orientation, the additivity and polarity of AV3 promoter were demonstrated. Comparative studies showed that AV3 promoter is fifteen-folds stronger than geminivirus Rep promoter, and equivalent or better than that of the well-studied bacterial constitutive promoter, rrnB P1. Expression of reporter-fusion proteins revealed that the putative translation start site of AV3 open reading frame (ORF) locates at nt 868, with a product of 8 amino acid in length. For practical applications, the novel promoter was utilized as part of a convenient cloning vector with an insertion-inactivation screening mechanism. A mutant with three- to five-fold increase in GFP expression was isolated in the construction process. By northern, western blot, nucleotide sequence analyses, and substitution experiments, it is revealed that a point-mutation at nt 885 of the vector is responsible for the difference in GFP expression levels. The results obtained from this study will further facilitate the understanding of gene regulation of geminivirus in prokaryotic systems and the development of geminivirus-based vectors.
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