【作者】 梁莹；

【导师】 冯家勋；

【作者基本信息】 广西大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 作者所在实验室以前工作表明含有水稻黄单胞菌水稻致病变种(以下简称Xoo)13751 DNA的重组质粒pGXN3000上至少有4个基因(rpfA、rpfB、rpfC、rpfF)能正向调控该菌的胞外多糖的产生及致病性并已完成了pGXN3000的序列测定。序列分析表明pGXN3000的rpfA上游有4个完整的ORFs，分别为pdeA、cheB、pin、pcaD。pdeA基因为2262 bp，可编码一个含754个氨基酸的产物，该产物与地毯草黄单胞菌柑橘致病变种(以下简称Xac)的环二鸟苷酸磷酸二酯酶A(c-di-GMP phosphodiesterase A)有96％的相似性和93％的相同性。cheB基因为1074 bp，可编码一个含358个氨基酸的产物，该产物与Xac的谷氨酸甲酯酶(glutamate methylesterase)的相似性和相同性均为67％。pcaD基因为813bp，可编码一个含271个氨基酸的产物，该产物与野油菜黄单胞菌野油菜致病变种(以下简称Xcc)的β-酮已二烯醇内酯水解酶(beta-ketoadipate enol-lactone hydrolase)有92％的相似性和85％的相同性。pin基因产物的功能域分析表明其第189-198个氨基酸为锌指结合域(zinc finger binding domain，ZnF_NFX域)。在rpfC基因下游有—ORF的产物与Xcc的属于双组分调控系统家族的调节蛋白RpfG有99％的相似性和96％的相同性。 为了了解Xoo pdeA、rpfG、cheB、pin、pcaD基因在该菌在水稻致病过广西大学硕_l:学位论文程中的作用和功能，我们用同源自杀质粒单位点整合突变方法分别构建得到了该菌的p认纳一突变体(GXNI 256)、，月一突变体(GXNI 258)、CheB一突变体(GXNI 255)、刀j厅突变体(GXN1269)和户eal)一突变体(GXN1261)。 水稻植株剪叶接种试验表明，xo口p/n-突变体GXNI 269在水稻上引起 的病斑长度与野生型13751的没有显著差别，说明xo口pl’n基因可能与其在 水稻上的致病性无关。无论是在高接种浓度(OD600二0.1)还是在低接种浓度 (0D600=0.001)下，而oFp几一突变体GXNI 258、p动纳一突变体GXNI 256和pCaD- 突变体GXNI 261在水稻上引起的病斑长度显著降低，在高接种浓度(OD600=0.1)下，GXNI 25a、GxNI 256和GxNI 261在水稻上引起的病斑长度分别是野 生型13751的21.37%、64.69%和77.44%，在低接种浓度(0D600=0.001)下 则分别是野生型13751的14.47%、67.99%和76.10%，说明而。的rP招、刀决斌和pCao基因与其在水稻上的致病性有关。 平板检测表明xo口p丈介绒一突变体GXNI 256、rP招一突变体GXNI 258的胞外多糖和OSF因子的产量均显著减少，说明xo口胞外多糖和OSF因子的产生与rP招和p尤论斌基因有关。xo口pde月基因产物的功能域分析表明其第317一488个氨基酸为具有环二鸟昔酸磷酸二醋酶A活性的OUFI域和第498一745个氨基酸为具有二鸟昔酸环化酶活性的DU「2域(也称GGOE「基序)，这两个酶可能参与调控细胞内信号分子环二鸟昔酸的浓度。xo口rP凡基因产物的功能域分析表明第1 92一329个氨基酸为HD域，属于依赖金属的磷酸水解酶家族。关于信号分子环二鸟昔酸与病原菌在植物上的致病的相关性目前还未见报道。平板检测表明xo口p决流基因能很好地恢复rP绍一突变体GxNI 258产Ds「的能力，xo口rP凡基因也能很好地恢复pdeA一突变体GXN 1 256产胞外多糖和DSF的能力。 广西大学硕士学位论文 水稻植株剪叶接种试验表明cheB一突变体GXN 1 255在水稻上引起的病斑长度与13751的没有显著差异，但是水稻植株喷雾接种试验表明cheB一突变体GXN 1 255在水稻上引起的病情指数是1 3751的48.91%。毛细管趋化应答试验表明ch出一突变体的趋化能力显著降低，说明cheB基因与xo口的趋化应答有关，xo。的ch出基因与其在水稻上的致病性有关可能是通过控制其趋向水孔有关。进一步说明趋化性可能在植物病原细菌致病的早期起重要作用。更多还原

【Abstract】 It was previously shown that a cosmid clone pGXNGOOO containing Xanthomonas oryzae pv. oryzae (here after Xoo for short) 13751 DNA had at last four genes (rpfA rpfB rpfC rpfF), mutation in any of these genes resulted in significant reduction in the production of EPS and virulence of this bacteria. The plasmid pGXN3000 had been sequenced previously and sequence analysis revealved that there are at least four ORFs (pdeA,cheB,pcaD and pin) at the upstream of rpfA gene. The pdeA gene was 2262 bp, encoding a protein with 754 amino acids. PdeA has 96% similarity and 93% identity with c-di-GMP phosphodiesterase A of Xanthomonas axonopodis pv. citri str. 306. (here after Xac) .The cheB gene was 1077 bp, encoding a protein with 358 amino acids. CheB has 67% similarity and 67% identity with glutamate methylesterase of Xac. The pcaD gene was 813 bp,encoding a protein with 271 amino acids.PcaD has 92% similarity and 85% identity to beta-ketoadipate enol-lactonehydrolase of Xac. The pin gene was 651 bp. Amino acid 189-198 of P i n formed z i nc f i nger b i nd i ng doma i n (ZnF-NFX doma i n). There is one ORF dowmstream of rpfC, which encodes a protein having 99% similarity and 96% identity with Xcc RpfG, a regulator protein of two-component regulatory systems.In order to understand the function of the above mentioned five genes in Xoo 13751, nonpolar mutant of 13751 in pdeA(GXN1256), cheB (GXN1255), pcaD (GXN1261), pin (GXN1269) or rpfG (GXN1258) was constructed by homologous suicide plasmid integration.The lesion length of rice leaves caused by Xoo pin-(GXN1269) mutant was similar to that caused by Xoo wild type strain 13751, showing Xoo pin may not be necessary for the virulence of Xoo on rice. The virulence of Xoo rpfG- (GXN1258) , pdeA-(GXN1256), pcaD-(GXN1261) was significantly reduced compared to Xoo wild type strain 13751 both at high inoculation and low inoculation concentrations. The lesion length of rice leaves caused by Xoo rpfG-(GXN1258) , pdeA(GXN1256)and pcaD- (GXN1261) was 21. 37%, 64. 69% and 77. 44% of that caused by Xoo wi Id type 13751 at high inoculation concentration, respectively, and was 14.47%,77.99% and 76.10% of that caused by 13751 at low inoculation concentration, respectively, indicating that XoopdeA, rpfG and pcaD genes are required for the full virulence of Xoo on r i ce.The analysis of Xoo PdeA revealed that its ami no acid 317-488 formed DUF1 domain with c-di-GMP phosphodiesterase A activity and ami no acid 498-745 formed DUF2 domain (also named GGDEF motif) with diguanylate cyclase activity. Diguanylate cyclase is involved in the sythesis of c-di-GMP and phosphodiesterase A is involved in the turnover of c-di-GMP. Xoo pdek can restore the production of DSF of rpfG-mutant(GXN1258) and Xoo rpfG can restore the production of EPS and DSF of pdek mutant (GXN1256).The lesion length of rice caused by Xoo cheB-(GXN1255) with leaf-clipping inoculation was similar to that caused by Xoo wi Id type, while the disease index caused by Xoo cheB-(GXN1255) on rice with leaf-spraying inoculation was 48.91% of that of 13751. The chemotactic respone assay revealed the ability of chemotactic respone of Xoo cheB-(GXN1255) was significantly reduced, indicating that cheB is involved in chemotaxis of 13751. The role of cheB in the virulence of Xoo may through controlling the ability of bacteria to move towards hydrothodes. Chemotaxis may play an important role at the early stages in pathogenesis of phytopathogenic bacteria.更多还原