| Summary: | 碩士 === 輔仁大學 === 生物學系 === 90 === A subtractive hybridization technique was employed to obtain DNA probe specific for Ralstonia solanacearum race 2. One cloned fragment hybridized under stringent conditions to DNA of race 2 strains, but not others race 1 strains. The clone was designated pS1.0k and then sequenced, amino acid sequence deduced from the nucleotide sequence showed homology of 59.6% to IS100 of Yersinia pestis. Further analysis of the region flanking this fragment showed structural features of a bacterial insertion sequence (IS) element. DNA sequence analysis indicated that this IS is 1956bp in length and delimited by two imperfect inverted repeats of 29bp with 8 mismatches. Besides, less conserved sequence elements, termed multiple terminal repeats, occur at both termini. Insertion of this IS into target site generate a direct repeat of 6bp. The G + C content of this IS is 64.37%. It consists of two adjacent open reading frames (orf), overlapping for 4bp. Nevertheless, two possible translational starts separated by seven codons were found in the orf1 gene. Therefore orf1 may encode for two polypeptides of 338 and 331 amino acids respectively. Both of the deduced amino acid sequence of ORF1 contains a conserved D-57-D-48-E motif and helix-turn-helix domain, whereas the protein of 262 amino acids deduced from ORF2 contains the A and B motifs of the NTP-binding site. These display characteristic features of members of IS21 family. Result of DNA homologies search by Fasta program in GCG showed that this element is having similarity of 39% to 58% to members of IS21 family. According to these characters and significant homology with similarly organized ORFs present in insertion sequences belonging to the IS21 family, this IS suggests to be a new member of IS21 family; hence, the name of ISRso19 was assigned by IS database (Genbank accession no. AF450275). Specific oligonucleotide primers were designed based on the internal nucleotide sequence of ISRso19. The PCR product of 683bp only can be amplified from Ralstonia solanacearum race 2 but neither in race 1 strains nor others phytopathogens. Since race 2 contain ISRso19 whereas strains in Taiwan (race 1 biovar 3 & 4) have its specific insert sequence, IS1405 which belong to IS5 family (Appl. Environ. Microbiol. 67 (2001): 3943—3950), thus by using multiplex PCR with these two IS specifically designed primers, race 2 can be distinguish from race 1. On the other hand, result of the IS database and Genbank searches displays other 24 types of Ralstonia solanacearum insertion sequence which are distribute between 7 IS family. Further investigation using PCR amplification with primers, which were designed based on the sequences of the 5’ and 3’non-coding regions of each IS5 family member, demonstrated that 5’ and 3’non-coding regions of IS are specific markers for each IS, and certain IS elements exist in certain bacteria. The results showed that IS was very useful for development of a specific and sensitive detection method for plant pathogenic bacteria.
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