十字花科黑腐病菌 rpf 基因串對鞭毛生成角色之探討

Characterization of the role of rpf cluster in flagellar biogenesis in Xanthomonas campestris pathovar campestris

張如珍(Ju-Chen Chang)

指導教授 ： 胡若梅

亞洲大學/健康學院/生物科技學系碩士班/碩士(2008年)

摘要

Xanthomonas campestris pathovar campestris (Xcc) 為革蘭氏陰性菌具單極性單鞭毛，會感染十字花科植物造成黑腐病。已知 Xcc 的胞外多醣（EPS）是重要的致病因子之一，而EPS的生合成會受 rpf gene 串的調控。本實驗室先前以插入突變的方法，將rpfC、rpfF、rpfG和rpfB基因破壞後加以試驗，結果顯示rpfF、rpfC和rpfG突變後，突變株即喪失致病能力以及降低EPS的產量。Xcc 之鞭毛是運動的工具之一。本研究目的在闡明rpf基因串對Xc之運動與其鞭毛基因在轉錄和轉譯階層之調控。
結果顯示，（1）在電子顯微鏡之觀察下rpfF和rpfC 突變後並無鞭毛生成；rpfG和rpfB突變後則仍有鞭毛；（2）溶菌斑測試結果顯示rpfF突變後對噬菌體（L7）有抵抗能力；（3）在轉錄階層， rpfC和rpfF基因正調控鞭毛基因 flgB、flgG、flhF、 fliC、fliQ、fliE和 fliL；rpfG 基因正調控鞭毛基因 flgG、flhF、 fliC、fliQ、fliE和 fliL；rpfB正調控鞭毛基因 flgB、fliC、fliQ 和 flhF 啟動子表現。（5）在轉譯階層rpfC正調控 FliC、FliA蛋白表現；rpfF正調控FlhF、FliA、FliC蛋白表現；rpfG正調控FliC及FliA 蛋白表現。

關鍵字

十字花科致病基因 ； 十字花科黑腐病 ； 科黑腐 ； 十字花 ； 病菌 ； 角色 ； 鞭毛 ； rpf 基因串

並列摘要

Xanthomonas campestris pathovar campestris (Xcc) is a Gram-negative bacterium and the causal agent of black rot disease in cruciferous plants. The exopolysaccharides (EPS) are one of the important virulence factors, whose biosynthesis is regulated by the rpf (regulation of pathogenicity factors) genes cluster. Mutants of rpfB、rpfF、rpfG and rpfC were constructed by insertional mutagenesis. It has previously been shown that rpfF、rpfC and rpfG mutants are avirulent and exhibit a decreased level of EPS. Nevertheless, a rpfB mutant displays little phenotypical change. Xcc is motile by using a single polar flagellum. In this work, experiments were carried out to elucidate the role of rpf genes on the bacterial motility as well as on the expression of flagellar genes in the transcriptional and translational level.
The results showed that, 1) electron microscopy demonstrated that rpfC and rpfF mutants have no flagellum and rpfB, rpfG mutants have single flagellum. 2) Spot test result demonstrated that a rpfF mutant become resistant to the infection of bacteriophage L7. 3) In the transcriptional level, rpfC、rpfG and rpfF genes positively control the expression of flagellar genes flgG、flhF、fliC、fliQ、fliE and fliL promoter activity. rpfB positively regulate the promoter activity of flgB、fliQ、fliC and flhF genes and rpfC、rpfF positively regulate the promoter activity of flgB. 4) In the translational level, rpfC positively regulates the expression of FliC and FliA; rpfF positively regulates the expression of FliA、FliC and FlhF; rpfG positively regulates the expression of FliC and FliA.

並列關鍵字

Xanthomonas campestris pathovar campestris ； the role of rpf cluster ； virulence genes

參考文獻

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