| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 91 === Abstract
To elucidate the regulatory mechanism of SOS response in Xanthomonas campestris pv. campestris (Xc17), activities of six DNA damage inducible promoters, recA, 2846-lexA, 5360-lexA, sulA, umuD and algU were analyzed. The promoter regions of these genes were cloned and fused to the luxAB gene from Vibrio fischeri. These promoter fusion luxAB reporter genes were then introduced into Xc17 chromosome by homologous recombination and identified by Southern blot. By measuring the luciferase activity, the transcriptional activities of these promoters in the presence or absence of mitomycin C were determined. Results of luciferase activity assay revealed that all the tested promoters except umuD promoter were DNA—damage inducible. The recA promoter was constitutively expressed during the MMC treatment and has 4 to 7 times higher activity when compared to that in the absence of mitomycin C. However, the 2846-lexA, 5360-lexA and sulA promoters could only be induced after longer exposure to MMC. To investigate the role of Xc17 lexA on the expression of recA, strains of recA-luxAB reporter gene in Xc17 2846- lexA- mutant and with 2846-lexA or 5360-lexA overexpression plasmid were constructed. Results showed that the induction ratio of recA promoter was decreased in 2846-lexA or 5360-lexA overexpression transformants. Although evidences suggested that Xc17 LexA can not specifically bind to recA promoter in vitro, the results indicated that lexA genes have an effect on the expression of recA gene. Moreover, the promoter activities of 2846-lexA and sulA were decreased in Xc17 2846-lexA- strain and the DNA damage induce expression of these promoters were at same level. Furthermore, in order to clone gene from Xc17 that allow expression of SOS response in E. coli, genomic DNA library of Xc17 was constructed on pSK- plasmid. The selection was carried out in E. coli ER1992 that contains the lacZ gene under the control of SOS—inducible dinD promoter. A total of 200 transformants that exhibited blue color on X-gal plate were identified and sequenced. Among these selected clones, four genes (ruvB、endonuclease V、groESL and xveII) were selected for further characterization. These four target genes were amplified and cloned into broad range host vector (pBBad22T) and then introduced into the Xc17 recA-luxAB reporter gene construct (XcRPlux). Results revealed that expression of recA promoter were increased three times when XveII and Endonuclease V were overexpressed. Moreover, by using two-dimensional gel electrophoresis analysis, the different expression patterns of Xc17 proteins in wild type and 2846-lexA- or recA- mutant were identified.
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