【作者】 杨万风；

【导师】 刘凤权；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 硕士

【摘要】 水稻黄单胞菌包含两个致病变种Xanthomonas orzae pv.oryzae(Xoo)和X.oryzae pv.oryzicola(Xooc)，在水稻上分别引起白叶枯病和细菌性条斑病。本研究对水稻黄单胞菌gacA、ppsA和zur基因进行克隆与鉴定，并对基因进行序列分析。用同源重组的方式构建相应基因的插入突变株，通过与野生型菌株进行表型比较来推知基因相应功能。 (一) 根据已公布基因组的X.axonopodis pv.citri(Xac)和X.campestris pv.campestris(Xcc)的gacA基因序列，设计简并引物GF／GR，采用PCR的方式从Xoo菌株PXO99和Xooc菌株RsGD42染色体中扩增了gacA基因的同源片断。核酸序列分析表明，gacA基因全长648bp，编码215个氨基酸的蛋白。Xoo和Xooc gacA基因只有2个碱基的差别。在与其它种gacA基因比较发现其在Xanthomonas属中是保守的。NCBI网站蛋白保守域搜索结果表明，GacA属于LuxR蛋白家族相似蛋白的一员。其具有一个CheB结构功能域，可能参与调控细菌的趋化性。 以引物XOR1／XOR2扩增gacA基因的内部片断，连接于PMD18-T，获得重组质粒pXOR和pXORs，电击转化质粒于野生型菌株电转感受态细胞中。通过单交叉同源重组的方式将重组质粒整合入基因组染色体中获得突变株。PCR和Southern杂交验证质粒插入的正确性。通过对0.1％Tryptone趋化性的调控来看，突变株较野生型的趋化性有了一定程度的下降。但对其它一些表型的测定结果证明，gacA基因并不是水稻黄单胞菌的全局性调控子。 (二)水稻白叶枯病菌基因组全序列已公布。通过基因组注释发现，Xoo只有一条糖异生路径，也就是苹果酸酶-磷酸烯醇式丙酮酸合成酶途径。本研究通过单交换同源重组的方式构建了磷酸烯醇式丙酮酸合成酶基因(ppsA)的非极性哭变体OSPAM。碳源利用检测显示，野生型菌株PXO99和互补菌株COSPAM能够在以柠檬酸、苹果酸、琥珀酸、延胡索酸、丙酮酸、甘油或乙酸作为唯一碳源的基本培养基上正常生长，而OSPAM则不能正常生长。说明PPSA途径是PXO99在糖异生过程中从四碳二羧酸合成磷酸烯醇式丙酮酸的唯一途径，这一结果和基因组注释相符。利用剪叶接种法接种感病寄主汕优63，突变体产生的病斑长度明显低于野生型。寄主叶内生长测定结果显示OSPAM的生长比PXO99低一个数量级。这些结果表明糖异生过程是白叶枯病菌致病及在寄主体内生长必需的。 (三)锌吸收调控蛋白(Zur)存在于许多细菌中，当细胞内的锌离子浓度过高更多还原

【Abstract】 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更多还原