| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 100 === 英文摘要
The Xan-D medium was used to isolate and differentiate Xanthomonas spp. from cruciferous seed. Xanthomonas spp. in Xan-D turned yellow-green, wet-shining and surrounded with a smaller milkly zone and a bigger clear zone colonies. The cabbage leaves inoculated with these Xanthomonas strains. The result showed that some leaves received “black rot disease” symptoms caused by Xanthomonas campestris pv. campestris (XCC) , some received “bacterial leaf spot” symptoms caused by Xanthomonas campestris pv. raphani (XCR), the other leaves didn’t get any disease as usual. The PCR amplification using the primer set, hrpF-PCR amplified a 424 bp DNA fragment for these XCC and XCR strains (called hrpF (+) strains) but non-pathogenic Xanthomonas strains (called hrpF(-) strains). Then, these hrpF (+) and hrpF (-) strains’ hrc genes which located on pathogenicity island was detected by PCR and southern hybridization. The result showed the hrpF (+) strains got pathogenicity island but the hrpF (-) strains didn’t. Thus, the hrpF-PCR is a useful method to differentiate pathogenic and non-pathogenic Xanthomonas spp. and detect the presence of pathogenicity island. The 16S rRNA sequence and the result of physiological and biochemical analysis of the hrpF (+) strains, X. campestris pv. campestris and X. campestris pv. raphani, were too similar to differentiate from each other. In this study, we accorded the mechanism of gene evolution to find a gene which can differentiate XCC and XCR by PCR amplification. We first analyzed the cellulase gene due to the ability of degrade plant cell wall is necessary for the plant pathogenic bacteria. Through the genomic DNA alignment, a cellulase gene, engXCA which is presence in most Xanthomonas spp. was found. Compared the phylogenic tree of engXCA to 16S rRNA, two trees showed the similar current, furthermore, the engXCA’s bootstrap value is much higher than 16S rRNA’s. That means the higher credibility of phylogenic tree of engXCA than 16S rRNA. Therefore we though the cellulase gene engXCA is an ortholog of xanthomonads. During the process of gene alignment, a paralog of engXCA formed by engXCA duplicated in XCC and XCR were found. The paralog of engXCA in XCC and XCR became two diverse genes, XCC3535 and XCR_3876. The sequences of XCC3535 and XCR_3876 were used to design specific primer sets, Xan-cel3535-F/ Xan-cel3535-R2 and XCR-cel3535-Fr/ Xan-cel3535-R2. Xan-cel3535-F/ Xan-cel3535-R2 amplified 1429 bp DNA fragments for XCC, and XCR-cel3535-Fr/ Xan-cel3535-R2 amplified 733 bp DNA fragments for XCR in PCR amplification . The multiplex PCR primer set combined two primer sets, differentiated XCC and XCR in once PCR analysis. Because of the pathogenicity of solanaceous of XCR, we wanted to differentiate other solanaceous pathogenic xanthomonads by engXCA’s paralog. To find the paralog of engXCA in the solanaceous xanthomonads, we did XCC3535 BlastP and the engXCA’s paralog in the Solanaceous pathogenic Xanthomonas spp., ,X. gardneri and X. vesicatoria were found. According to those two diverse gene design two primer sets, XG-cel3535-F/ Xan-cel3535-R2 amplifying 423 bp DNA fragment and XV-cel3535-F/ Xan-cel3535-R2 amplifying 1642 bp DNA fragment in PCR amplification, used to classify the solanaceous pathogenic Xanthomonas spp.
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