【作者】 赖洁玲；

【导师】 何勇强； 唐东阶；

【作者基本信息】 广西大学 ， 微生物学， 2008， 硕士

【摘要】 在细菌中,锌吸收调控蛋白Zur(zinc uptake regulator)(由zur基因编码)的主要功能是当细胞内锌离子浓度过高时抑制锌吸收系统的表达。但是,本实验室的研究表明,在十字花科黑腐病菌(Xanthomonascampestris pv.campestris)中,zur(基因编号为XC1430)除了调控锌离子平衡外,还参与了该菌的致病过程和EPS产生。在完成zur基因的功能鉴定后,本实验室还对zur基因的启动子进行了初步研究,推测zur基因可能存在两个独立的启动子P1(基因组注释的ATG前面114 bp)和P2(基因组注释的zur基因读码框内,ATG下游63 bp处的另一个翻译起始密码子GTG前108 bp)。为了证实这一假设,本研究分别构建了启动子P1和P2与无启动子gusA基因的融合报告质粒pL6PlgusA和pL6P2gusA,并检测它们在野生型菌株8004中的GUS活性,结果表明,P1和P2均有启动子活性;进一步的实验研究结果表明P1和P2的表达均不受锌离子的影响。为了确定P1和P2的转录起始位点,我们进行了5′-RACE实验,结果表明在ATG上游23 bp处的A碱基是P1的转录起始位点,但是在我们所用的实验条件下没有检测到P2的转录起始位点。由于GUS活性结果表明P1和P2都具有启动子活性,因此我们推测zur既可以从ATG起始也可以从GTG起始翻译,分别编码两种Zur蛋白即ZurL(从ATG起始)和ZurS(从GTG起始)。为了证实这一点,我们分别构建了带有翻译起始密码子ATG→ATA和GTG→GTA点突变的zur基因的质粒pL6zurATG和pL6zurGTG并分别导入zur的缺失突变体中,然后对点突变互补菌进行锌敏感性、EPS产量以及致病性进行检测。结果发现,将GTG点突变后(只能合成ZurL蛋白),重组质粒pL6zurATG可以互补zur突变体的锌敏感、致病力和EPS合成表型;而将ATG点突变后(只能合成ZurS蛋白),重组质粒pL6zurGTG可以互补突变体的致病力、EPS合成而不能互补其锌敏感表型,表明从ATG起始或从GTG起始编码的蛋白均有生物学功能,且在功能有交叉也有差异:从ATG起始编码的蛋白ZurL具有锌平衡、EPS合成、致病调控功能,而从GTG起始编码的蛋白ZurS,只有EPS合成、致病调控功能而没有锌平衡功能。为了进一步鉴定ZurL和ZurS蛋白是否存在,我们在NYGB培养条件下对点突变互补菌株和缺失突变体菌株的Zur蛋白进行了Western blotting检测,结果发现在点突变体菌株的细胞提取物均能检测到一条杂交带,而在缺失突变体菌株的细胞提取物中未能检测到杂交带,其结果表明在各自的点突变互补菌株中ZurL和ZurS是存在的,其结果与点突变体的表型检测结果相符。但Western blotting检测野生型菌株Xcc 8004的细胞提取物时,未能检测到两条杂交带。以上这些结果证明,(1)除了ATG前面的启动子(P1)外,zur基因读码框内GTG上游-108 bp区域(P2)具有启动子活性;(2)从ATG起始编码的蛋白ZurL具有锌平衡、EPS合成、致病调控功能,而从GTG起始编码的蛋白ZurS,只有EPS合成、致病调控功能而没有锌平衡功能。但在所用的实验条件下,用Western blotting未能检测到Xcc 8004细胞内有两种Zur蛋白。更多还原

【Abstract】 In bacteria,the zinc uptake regulator Zur is encoded by zur gene and typically functions as a repressor to repress the expression of zinc uptake systems when the zinc concentration inside the cell reached a threshold. However,our previouse work showed that,in Xanthomonas campestris pv.campestris(Xcc),in addition to involved in zinc homeiostasis,zur (ORF number XC1430)also play an important role in pathogenesis and EPS production of this pathogen.Furthermore,our unpublished data suggested that Xcc zur may contain two independent promoters,P1, which is located upstream of the annotated start coden ATG,and P2, which is upstream of GTG located inside the coding region ofzur(63 bp downstream of ATG).To confirm this hypothesis,in this study,we constructed the promoter(P1 and P2)-gusA transcriptional fusion reporter plasmids pL6PlgusA and pL6P2gusA,and tested their GUS activity.The result showed that both P1 and P2 displayed promoter activity,and expression of P1 and P2 is not affected by zinc concentration.In order to identify the transcriptional start site of P1 and P2,we carried out the 5’-RACE analysis and the result show that the A base located 23 bp upstream of the annotated start coden ATG is the transcriptional start site for P1,but we can’t detect the transcriptional start site of P2 under the experimental conditions.Based on the fact that P1 and P2 are functional promoters,we speculated that the two promoters can lead to the sythesis of two different functional Zur proteins,one(designated as ZurL)was translated from the ATG and the other(designated as ZurS)was translated from GTG.To confirm this,the 2 start codens,ATG and GTG,were respectively mutated(ATG→ATA,GTG→GTA)by site-directed mutagenesis,and the function of the mutated zur were assay by complementation of the zur deletion mutant.Result showed that,the plasmid pL6zurATG which expresses ZurL can restore all of the phenotypes(sensitive to high zinc concentration、reduced virulence and EPS production)of the zur mutant, while the plasmid pL6zurGTG which expresses the ZurS can restore EPS production and virulence but not high zinc sensitivity,indicating that ZurL and ZurS have overlap and different functions:ZurL has zinc homeostasis regulation,EPS production and virulence function ZurS has EPS production and virulence function.To test if the ZurL and ZurS do exist in Xcc cell,we performed Western blotting analysis and the result show that one hybrid band was observed in the cell extractes of each one site-directed complement strain,but only one hybrid band was observed in the cell extractes of the wild type strain 8004.Based on the above results,we conclude that:(1)it is very likely that Xcc zur has two different promoters:except for the promoter upstream of ATG(P1),the region upstream of GTG is also a functional promoter(P2).(2)ZurL(translated from ATG)and ZurS(translated from GTG)have overlap and different function.更多还原