| Summary: | 碩士 === 國立臺灣大學 === 植物病理學研究所 === 90 === A random (”shotgun”) sequence strategy was conducted to investigate the genome of the phytoplasma associated with peanut witches’ broom (PNWB) in this study. Total DNA of periwinkle infected with PNWB phytoplasma and that of healthy periwinkle were used as a mode of infected periwinkle probes for differential screening of lambda ZapII genomic libriay and pBluescriptII SK（-）recombinant plasmids of PNWB-Phytoplasma. Eight out of 108 random clones thus selected were completely sequenced and analyzed. A total of 29 putative open reading frames (ORFs) were identified among the sequences. The insert DNA of recombinant plasmid C2 consists of 4225 nucleotides and encompasses an incomplete ORF homologous to extrachromosomal DNA of other organisms. In order to clone the whole ORF and to analyze the recombinant plasmid C2 further, a pair of oligo-nucleotides primers C2R1/C2F1 for inverse polymerase chain reaction (IPCR) were designed according to the nucleic acid sequences of the recombinant plasmid C2. Total DNA from diseased periwinkles infected with PNWB phytoplasma was prepared as the template for IPCR reaction. A 265 bp fragment was amplified using the primers C2R1 and C2F1. The result indicates that the 4.2 kbp insert DNA of the recombinant plasmid C2 is a circular form DNA (plasmid). The IPCR product was sequenced and aligned with the recombinant plasmid C2 and its nucleic acid sequence is consistent with that of recombinant plasmid C2, and thus a complete ORF was determined. According to the analyses of nucleic acid and amino acid sequences, this ORF was identified to be a putative Rep gene. The results of Southern hybridization analysis using Rep gene as a probe suggested that multiple copies of Rep gene may exist in PNWB-Phytoplasma. Recombinant plasmid H13 encompasses a complete ORF of 3988 nucleotides. The sequence of this ORF is homologous to the transposase gene of other organisms. An eight-nucleotide inverted repeats between nucleotides 1713-1720 and 2737-2744 near the ORF was found and a conserved DDE motif in this ORF was identified. According to the results of the Southern hybridization analysis by using the probe for transposase gene or insertion sequence element, it is suggested that only one copy of transposase and insertion sequence element may exist in PNWB-phytoplasma.
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