| Summary: | 碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 106 === The drug resistant bacteria in the environment highly threaten human health. Currently, the river’s ecosystem evaluation method does not contain bacterial drug resistance testing. Therefore, we are not familiar with the drug resistance bacteria in the water environment. This study is based on samples from 13 different areas from the rivers in Taichung including Liuchuan Canal, Luchuan Canal, Meichuan Canal, Mayuanton River, and Hanxi River, to isolate and identify multidrug resistance bacterial strain including Aeromonas caviae, Aeromonas hydrophila, Aeromonassobria, Aeromonas spp., Escherichia coli, Klebsiella pneumoniaandProvidencia stuartiiwhich resist ampicillin(Ap), kanamycin(Km)and tetracycline(Tc) these three kinds of antibiotic. There was only one sample that does not have multidrug resistantbacteria. However, the other 12 samples contained 1~3 multidrug resistance bacterial strains. This shows that these rivers are seriously polluted. The electropherogram of the plasmid we extract from the bacterial strain shows that even same culture in different water environment, the plasmid inside has huge differences. Moreover, some bacterial strains does not carry plasmid but have drug resistance. These bacterial strains’ drug resistance gene might be on the chromosome. Among all, an E. coli isolated from the middle reach of Mayuanton River has a little plasmid about 3.0 kb is named pEco3K. By cloning and PCR amplification strategy sequencing this plasmid, the total length of pEco3K plasmid nucleic acids sequence is 2,934bp, and average of G+C is 50.0%. The result of comparison between nucleic acids sequence shows that pEco3K plasmid does not carry drug resistance gene. There are 7 ORFs are hypothetical protein that contains ColE1 origin of replication’s (oriV) conserved sequences, and similar to ColE1 plasmid origin of replication primer “RNA II”sequence and regulatory RNA II’s " RNA I " sequence. Cosmid the Gmrcassette fragment withpEco3K 1.0 kb BamHI fragment to become pLTG.pLTG has high stability and extensive hostility which can live and transfer in the host of Enterobacteriaceae. It is estimated to copy number 40~60. pLTG and pOK12 are compatible that could exist in the same cell. The result of the study shows pEco3K could be developed into vector for gene cloning. The most important thing is that in the environment, pEco3K could horizontal transfer to different bacterial strain by its tiny and stable existence for cloning, which plays an importantrole in horizontal gene transfer.
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