【作者】 何飞燕；

【导师】 唐纪良； 何勇强；

【作者基本信息】 广西大学 ， 微生物学， 2004， 硕士

【摘要】 野油菜黄单胞菌野菜致病变种(Xanthomonas campestris pv．campestris，以下简称Xcc)是一种能在全球范围内引起十字花科植物黑腐病的病原性细菌。胞外多糖是Xcc的重要致病因子之一，也是一种重要的工业多糖。本实验室已构建了Xcc 8004的Tn5gusA5插入突变库，并对16462株突变体的EPS产量进行了平板检测，获得了75株胞外多糖产量下降突变株。本工作用2％葡萄糖的NYGA平板上检测，对这75个突变体进行了重新筛选，得到了51株胞外多糖缺陷的突变体，它们分布在45个ORF中。其中11个ORF前人已证实与胞外多糖合成有关：34个ORF未报道与胞外多糖的产生相关，这34个ORF中13个编码的为假设保守蛋白。本工作选择这13个编码的为假设保守蛋白的基因的一个，编号为XC0877，进行进一步研究，以确定该ORF是否与EPS合成有相关。根据Xcc 8004的Tn5gusA5插入突变库的资料，XC0877 ORF中有四个独立的Tn5gusA5插入突变体，其中有两个突变体为胞外多糖缺陷型，另两个突变体为胞外多糖产生正常。我们初步认为这种表型不一致的原因有可能是ORF界定有误。为了确证XC0877 ORF与胞外多糖产生的关系，我们采用同源单交换的方法构建了该ORF的非极性定点整合突变体。并对所得突变体进行了胞外多糖产量检测，出乎意料的是在这些突变体中大约有一半表现为缺陷型而另一半则表现为胞外多糖产生正常。用PCR方法扩增野生型XC0877 ORF(包括启动子)，并克隆到pLAFR3上，进行反式互补，结果未能恢复突变体的胞外多糖的产量。综合以上结果，XC0877 ORF是否与胞外多糖产生有关还需进一步研究。更多还原

【Abstract】 Xanthomonas campestris pv. campestris (Xcc) is an important pathogen of cruciferous plants, it causes the black rot disease of cruciferous crops worldwide. One of the products of X. campestris is excellular polysacchride(EPS) named xanthan gum. EPS is currently regarded as a pathogenicity factor during interaction between Xcc and host plants. In addition, EPS is also a sort of important polysacchride in industry. The Xcc 8004 mutant library was constructed by the insertion of transposition Tn5gusA5 in the laboratory. The EPS production of 16462 Tn5gwsA5 inserted mutants Xcc 8004 strains were tested and 75 had a fall in EPS production. In this study, the NYGA containing 2% glucose is used to re-select these 75 mutants. The result of re-selecting is 51 strains of EPS production deficiency were obtained. They are distributed in 45 ORFs. Among them, 11 have been verified to be relevant to EPS, and 34 have not been reported to have relationship with EPS. Furthermore, 13 of these 34 ORFs encode conserved hypothetical protein. XC0877 is one of these 13 ORFs and it is used to make a further study in order to verify whether it is relevant to the EPS synthesizing. According to the information of Tn5gusA5 5inserted mutants library, there are 4 independent Tn5gwsA5 inserted mutants in XC0877 in which 2 of them are deficient in EPS production and the other 2 are normal in EPS production. Thereason why there are two phenotypes is thought initiatively as a fault of ORFs defining. In order to prove that XC0877 is related to EPS production, we construct the XC0877 non-polar site-specific insertion mutant. It is found that nearly half of these mutants are deficient in EPS production and the other half is normal in EPS production. Using the way of PCR to amplify wild type XC0877 (including promoter) and then clone it on pLAFR3 to undergo complemtation in trans, the result is that the EPS production of the mutant dose not recover. According to all the results above, whether XC0877 is relevant to EPS production is needed to do further research.更多还原