碩士 / 國立臺灣大學 / 植物學系 / 81 / The recent application of nucleic acid probes to detect bac-
teria found in environmental samples showsgreat promise. The
ap- plications of preexcisting DNA hybridization techniques in
deter- mining populationsin environmental samples were
investigated. In this study, first we use shrimp pathogenic
bacterium Vibrio harveyi as tested organism. Since this
organism is a luminescent bacterium and the structural genes
controlling of luminescent system has been studied and
cloned to several plasmids. Having obtained a plasmid
containing Lux A and Lux B genes, We have transformed the
plasmid pBR322 into Escherichia coli RR1. After growing in LB
broth, the plasmid was isolated and cut by restric- tion
endonuclease HindIII to prepare probe. Colony hybridization
experiment ws performed by theprobe with Vibrio spp.. However
the specificity of Lux gene was not good for V. harveyi. We
also use fish pathogenic bacteria Vibrio alginolyticus and
Vibrio anguillarum as tested organism. Randomly cloned
fragments of DNA from V. alginolyticus and V. anguillarum
were used as colony hybridization probes for Vibrio spp. and
other bacteria. Restriction endonuclease HindIII and EcoRI
digestion fragments of DNA from V. alginolyticus and V.
anguillarum were inserted into vector pUP19. Purified
recombinant DNA from host cell E. coli TG1 were digested by
same restriction endonuclease and subjected to
electrophoresis on 1﹪ agarose gel. Among 0.5 K pb∼ 2 K pb
size of insered DNA were recovered from agarose gel with DEAE
membrane to prepare non-radioactive Dig-labeled probes. Nine
out of 102 cloned fragments could hybridize only to V.
alginoly- ticus and 14 outof 94 cloned fragments only to V.
anguillarum.
| Identifer | oai:union.ndltd.org:TW/081NTU00366002 |
| Date | January 1993 |
| Creators | Shieh,Yu-Ling, 謝玉玲 |
| Contributors | Tsay,San-San, 蔡珊珊 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 74 |
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