| Summary: | 碩士 === 國立中央大學 === 生命科學研究所 === 87 === Abstract
Using insert DNA fragment of clone 138, which contained Mycoplasma genitalium rDNA gene, as probe to screen theλZAP/EcoRI genomic library of loofah witches' broom (LfWB) phytoplasma, 8 clones were obtained. Thy were clone 3-1, 3-2, 3-3, 3-4, 4-1, 4-2, 4-3, and clone 4-4. The clone 4-2 contained part of 16S rDNA, 16S-23S internal transceibed spacer (ITS), and first one-third of 23S rDNA. The other 7 clones were converted into phagemid by in vivo excision, and 4 colonies were obtained: clone Y2, Y3, Y5, and Y8.
The complete nucleotide sequences of clone Y3 and clone Y5 were determined and compared to Mycoplasma genitalium genomic DNA databank. It revealed that the clone Y5 contains protein B gene, protein C gene, and part of protein D gene of oligopeptide ABC transporter permease, and the clone Y3 contained an impact thymidylate kinase (tmk) gene. The opening reading frame of tmk, from nucleotides 1,206 to 1,835 encoded a protein of 209 amino acids with a molecular weight of 24,791 Da. This ORF was preceded by a strong consensus sequence for ribosomal binding site (rbs) and the putative -10 and -35 regions. The protein contains three conserved motifs of TMK. The deduced amino acid sequence of tmk was compared with previously determined sequence of several cellular and viral TMKs. The TMK of LfWB was more similar to prokaryotic TMK. The result of Southern hybridization analysis indicated that there was only a single copy of tmk gene in phytoplasma genome.
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