Cloning And Fuctional Study Of GacA, PpsA And Zur Genes Of Xanthomonas Oryzae

Xanthomonas oryzae pv. oryzae(Xoo) and X. oryzae pv. oryzicola (Xooc) cause leaf blight and leaf streak on rice respectively. In this study, the gacA, ppsA and zur gene were cloned and sequence analyzed from X. oryzae, and the disruption mutant of the gacA , ppsA and zur gene were generated by single cross-over event. Compared with the wild type strain, the function of the gacA, ppsA and zur gene was speculated.(I) A gacA homologue, was cloned from the genomic DNA of X. oryzae, the bacterium that cause leaf blight and leaf streak of rice, with degenerated primers GF/GR by polymerase amplification reaction according to the homologous gacA sequence of X. axonopodis pv. citri(Xac) and X. campestris pv. campestris(Xcc). Sequence analysis showed that the 648 bp gacA gene in this study putatively encoded a 215 ammonia acid protein. Sequence comparison indicated there are 2 bases difference of gacA gene between Xoo and Xooc. Compared with other bacterium, we found that the gacA gene was conserved in the Xanthomonas genus. NCBI blast search indicated that GacA had a similar structure to that of other GacA proteins, and had conserved domain of LuxR-like proteins. Besides the structure, there is a similar CheB structure domain, it may regulate chemotaxis of the bacterium.The inner fragment of the gacA gene was generated by the primers of XOR1/XOR2, and ligated into PMD18-T. This resulted in new recombinant plasmids pXOR and pXORs. Subsequently, the plasmid was electroporated into Xoo PXO99 and Xooc RsGD42 respectively. Transformants presumably arising from a single cross-over recombination event between the plasmid and the genome of Xoo and Xooc were selected as putative mutant. The putative mutants were validated by PCR and Southern blot. Chemotaxis to 0.1% tryptone of the mutants was reduced compared to the wild type strains . However, we found that GacA was not the global regulator like a few Pseudomonas bacterium based on other results.(II) The genome sequences of X.oryzae pv. oryzae had publicized. We demonstrated genetically that X. oryzae pv. oryzae possesses only the PPS route in gluconeogenesis. To identify the function of the PPS route in gluconeogenesis in Xoo PXO99, the ppsA gene...