Application of the vir gene cluster to develop a molecular detection method for Xanthomonas spp.
| Autor: | Ping-Chen Wu, 吳秉蓁 |
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| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 英文摘要 The study of the vagA gene of Xanthomonas axonopodis pv. citri XCI3-1 leads to a discovery of a new gene, it is named vagX, whose deduced amino acid sequence shows 12-20% identity and 28-34% similarity to VirB8 homolog of Bordetella pertussis, channel protein VirB8 homolog of Brucella melitensis, VirB8 of Agrobacterium tumefaciens, and VirB8 type IV secretion protein of Sinorhizobium meliloti. Those proteins contain conserved region at C- and N- terminal. We supposed that vagX is a virB8-like gene，so renamed it as XvirB8. We designed a PCR primer pair, based on the XvirB8 gene. The PCR primer pair is specific to the genus Xanthomonas. Southern hybridization revealed that XvirB8FR probe detects all of the Xanthomonas tested. All of other phytopathogens tested are not detected, it says that XvirB8FR is specific to the genus Xanthomonas. Based on published genome sequences of X. axonopodis pv. citri strain 306 and X. campestris pv. campestris strain ATCC33913, Xanthomonas spp. contain one virB gene cluster including virB8, virB9, virB10, virB11, virB1, virB2, virB3, and virB4 genes. The variety and arrangement of genes in vir gene cluster of Xanthomonas are different from other pathogens. Numerous bacterial pathogens use type IV secretion systems ( T4SS ) to deliver virulence factors directly to the cytoplasm of plant, animal, and human host cells. We infer that Xanthomonas is related to type IV secretion/transport systems and vir gene cluster is related to pathogenicity. We designed PCR primer pairs, based on the vir gene cluster sequence in order to identify genus Xanthomonas. Designing two primer pairs, based on virB8 and virB1 gene. Using the XvirB8 and XvirB1 primer mixture to amplify，can amplify specific DNA fragments only from Xanthomonas spp. but not from other plant pathogenic bacteria. The D4B8 primer from vir gene cluster also amplify specific DNA fragments from all of Xanthomonas spp. tested. Aspect of identification of Xanthomonas species, the non-coding regions between virD4 and virB8 have significant difference between X. axonopodis, X. arboricola and X. campestris. Designing four species-specific primers according to the non-coding region in order to distinguish three species of genus Xanthomonas. Results of PCR reveal that four primers amplify spcific fragment of their specific species. By the neighbor joining method, phylogenetic tree analysis of virD4-virB8 intergenic region sequences reveal that Xanthomonas spp. tested divide into four groups. Furthermore, utilize the DNA sequences of XAC2613 between virB4 and virB6 of X. axonopodis pv. citri 306 to design a primer pair, XAC2613 primer. XAC2613 primer can amplify only X. axonopodis pv. citri, this primer pair is specific to pathovar citri. Accordingly, vir genes are very useful for identification and detection of plant pathogenic Xanthomonas spp. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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