| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 95 === Aeromonas hydrophila is pathogenic to fish and causes hemorrhagic lesions in gastric tract, liver, spleen and kidneys, and fatal hemorrhagic septicemia. We amplified the aroA gene of Aeromonas hydrophila isolated from carp by using polymerase chain reaction ( PCR ). The amplified product is 984 bps. It has 91.6％ homology to the aroA gene of Aeromonas hydrophila ATCC 7966T and 97.2％ homology of the amino acid sequence. We designed two pairs of specific primers to amplify the 5’ and 3’ region of the aroA gene. The two amplificons were C1 and C2, respectively. The C1 fragment was ligated with pEGFP-N1 digested with Sal I and Afl II to generate pAROAC1. And then, the C2 fragment was ligated with pAROAC1 digested with Ase I and Xho I to generate pAROAC2. In order to delete the pUC ori fragment, the pAROAC2 were digested with Ase I and Dra I and treated with klenow fragment and religated to generate pAROAC3. The RAK fragment was amplified from the pAROAC3 by PCR using primers C1-F and C2-R. The RAK fragment was cloned into pCR®II-TOPO□ to generate pTOPO-RAK. The pTOPO-RAK was digested with BstX I to generate TRB fragment. Allelic replacement of the Aeromonas hydrophila wild type isolated from carp with the TRB fragment was accomplished by homologous recombination. Selection of kanamycine resistant transformants and the auxotrophic deficency bacteria A. hydrophila TRB was obtained. It was confirmed phenotypically, owing to the absence of the aroA gene, by requiring supplementation with aromatic metabolites for growth in M9 minimal media. And it was confirmed genotypically by PCR.
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