| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 91 === The study of the location of IS1404 in Xanthomonas campestris pv. campestris (XCC) leads to the interrupted vagA gene (virulence-associated gene; AF360374), whose deduced amino acid sequence shows homology VirD4-like protein range from 19 % to 27 % and TraG/TraD family range from 19~31 %. Those proteins are coupling protein of type IV secretion mechanism, containing consensus Walker motif A and B. VagA gene belongs to virD4 homolog. Excluding XCC1-1 vagA (interrupted IS1404, IS1478), XCC1-1 also contains another virD4 homolog- vag2A, XCC33 contains another virD4 homolog- vagA. It is clear that in XCC have been discovered at least three different types of virD4 homologs including vagA (NTPase), vag2A and virD4, comparing with the XCC ATCC 33913 genome sequence project, those deduced amino acid displayed a high degree of identity 68~89 %. The 66 kDa VirD4 protein was produced in E. coli pET system. Although most VirD4 were insoluble and formed an inclusion body, a few soluble VirD4 still can be purified, the 64.3 kDa soluble VagADN30 was also be induced and purified. The purified soluble VirD4 and VagADN30 both have an ATPase activity. Using Southern hybridization assays hybridization with various probes shows XCC33ΩTc-4 is vagA double crossing over mutation. Besides, the virD4 gene single crossing over knock out mutant strain of XCC35 was obtained by a marker-exchange method. It was found that the rate of symptom progress was later than that of wild-type after inoculation vagA knock out mutant and wild-type strains into cabbage, respectively. But, the rate of symptom of XCC35ΩTc was faster than of wild-type. The results indicated that the vagA and virD4 were involved in the virulence of XCC, respectively. When an internal fragment of virD4 homolog was used as a probe, the virD4 homologs are widely distributed in Xanthomonas phytopathogenic bacteria including three species: campestris, axonopodis and arboricola, except the X. oryzae, moreover, the virD4 homologs do not exist in other phytopathogenic bacteria such as Pseudomonas, Ralstonia, Burkholderia and Erwinia. Further three sets of PCR oligonucleotide primers distinguishing three types of virD4 homologs, revealed that all tested forty-one strains of Xanthomonas bacteria contain virD4, but some also have another virD4 homolog like vag2A or vagA. The results showed that in Xanthomonas including four species the existence of virD4 homologs can be group into five types. VirD4 specific primer can amplify a 0.4 kb PCR product from all forty-one Xanthomonas bacteria, but not from other plant bacteria in a PCR reaction. Therefore, the primer set is Xanthomonas-specific and can be used to identify Xanthomonas phytopathogenic bacteria. Further analysis of the existence of virD4 homologs in different strains of XCC, PCR and Southern hybridization analyses revealed the mrdB gene is located in the upstream of vagA gene; the situation of insertion sequence in vagA gene was determined by PCR reaction. Cloning and sequence analyzing revealed that the types of insertion sequence including IS1404, ISxcC1 and IS1478, and the interrupted location of different IS was also different, the interrupted location of IS in XCC12 group was outside of vagA coding region. And the virB8 gene is located in the downstream of virD4 gene, determined by PCR reaction. Using LA PCR and Southern hybridization assays revealed the XCC33 group not only having vagA but also virD4. These results showed eighteen strains of XCC could be divided into six groups on the basis of existence of virD4 homologs and insertion sequence. Analysis the XCC ATCC 33913 and X. axonopodis pv. citri str. 306 genome sequence present the array of mrdB-vagA-XCC3461-XCC3460-peh-1 in XCC ATCC 33913 and the array of mrdB-peh-1 in X. axonopodis pv. citri str. 306 was obvious different. When the internal fragment was used as PCR primer set or probe, it revealed that XCC3461 gene is limited to XCC and XP195; the peh-1 gene is widely distributed in all tested Xanthomonas. Furthermore, the array in XCC and XP195 were of mrdB-vagA-XCC3461-XCC3460-peh-1, in X. axonopodis, X. campestris pv. mangiferaeindicae str. XCM29-3, XCM32-1, X. arboricola and X. oryzae were mrdB-peh-1. Based on the vag2B and upstream of vag2A DNA fragment were used as a probe, examining the forty-five strains of Xanthomonas, it showed that they do not have obvious association. In conclusion, a model is proposed that the vagA and virD4 may be acquired by two distinct horizontal gene transfers, later some IS interrupted in the vagA region and the vag2A may be the gene duplication from the vagA gene.
|
|---|
