Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92234
標題: Application of Xanthomonas campestris pv. campestris bla promoter
十字花科蔬菜黑腐病菌的 bla 啟動子之應用
作者: 李佩真
Pei-Zhen Li
關鍵字: bla promoterro;bla 啟動子
引用: 陳駿義 (1998). Xamthomonas campestris pv. campestris β-lactamase 基因之選殖與定序. 國立中興大學分子生物研究所碩士論文. 陳志宏 (1999). 十字花科蔬菜黑腐病菌β-lactamase 基因之選殖與定序. 國立中興大學分子生物研究所碩士論文. 陳義元 (2001). 十字花科蔬菜黑腐病菌β-lactamase 基因之功能與特性分析. 國立中興大學分子生物研究所碩士論文. 李佳霓 (2006). Xamthomonas 溶裂型噬菌體 phiL7 與 phiXo411之探討. 國立中興大學分子生物研究所碩士論文. 陳巨威 (2009). 十字花科蔬菜黑腐病菌β-lactamase表現之調控. 國立中興大學分子生物研究所碩士論文. 黃崧銘(2010). 多元轉錄調控因子 Clp對 Xamthomonas campestris pv. campestris β-lactamase 基因之訊號序列與運送系統之探討. 國立中興大學分子生物研究所碩士論文. 蔡任濠 (2010). 噬菌體 phiL7 溶裂基因串之功能探討. 國立中興大學分子生物研究所碩士論文. 鄭宜寧 (2012). 以植物誘導型啟動子表現溶裂基因的模式系統之建立. 國立中興大學分子生物研究所碩士論文. 莊明芬 (2012). 以植物誘導型啟動子表現外源基因的重組噬菌體之構築. 國立中興大學分子生物研究所碩士論文. 謝宗佑(2012). 以 E.coli 表現系統大量表現抗菌胜肽 Epinecidin-1 及Lactoferricin B並評估其有效性及安全性. 國立中興大學分子生物研究所碩士論文. 鄧福勝 (2012). Xamthomonas campestris pv. campestris β-lactamase 的運送與表現. 國立中興大學分子生物研究所碩士論文. Asgarali, A. K., A. Stubbs, A. Oliver, D. J. Vocadlo, and B.L. Mark. 2009. Inactication of the glycoside hydrolase NagZ attenuates antipseudomonal beta-lactam resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 53:2274-82. Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M. 1992. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol. 73:472-9. Balcewich, M. D., T. M. Reeve, E. A. Orlikow, L. J. Donald, D. J. Vocadlo, and B. L. Mark. 2010. Crystal structure of the AmpR effector binding domain provides insight into the molecular regulation of inducible ampC beta-lactamase. J Mol Biol 12:17-41 Chen H.L., Yen C.C., Lu C.Y., Yu C.H., Chen C.M. 2006. Synthetic porcine lactoferricin with a 20-residue peptide exhibits antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. J Agric Food Chem 54:3277-82. Daniels, M. J., C. E. Barber, P. C. Turner, M. K. Sawczyc, R. J. Byrde, and A. H. Fielding. 1984. Cloning of genes involved in pathogenicity of Xamthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. EMBO J 3:3323-8. de Creey-Lagard, V., Glaser, P., Lejeune, P., Sismeiro, O., Barber, C. E., Daniels, M. J., and Danchin, A. 1990. A Xamthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity. J Bacteriol 172:5877-5883. Ellison, R. T., 3rd and T. J. Giehl. 1991. Killing of gram-negative bacteria by lactoferrin and lysozyme. The Journal of clinical investigation 88:1080-1091. Eisenstark, A. 1967. Bacteriophage Techniques. Methods in Virology 1:449-524 Goldsmith, M. E. & W. H. Konigsberg. 1997 Adsorption protein of the bacteriophage fd: isolation, molecular properties, and location in the virus. Biochemistry 16: 2686-2694. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 166:557-80. Hanson, N. D., and C. C. Sanders. 1999. Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae. Curr Pharm Des 5:881-94. He, Y. W., Ng, A. Y., Xu, M., Lin, K., Wang, L. H., Dong, Y. H., and Zhang, L. H. 2007. Xamthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Mol Microbiol 64:281-292. Naidu, S. S., U. Svensson, A. R. Kishore and A. S. Naidu. 1993. Relationship between antibacterial activity and porin binding of lactoferrin in Escherichia coli and Salmonella typhimurium. Antimicrob Agents Chemother 37:240-245. Sukchawalit R., Vattanaviboon P., Sallabhan R, Mongkolsuk S. 1999. Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas. FEMS Microbiol Lett. 181:217-23. Tseng, Y.H., Choy, K.T., Hung, C.H., Lin, N.T., Liu, J.Y., Lou, C.H., Yang, B.Y., Wen, F.S., Weng, S.F., Wu, J.R. 1999. Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J. Bacteriol. 181:1 117-125 Tseng Y.H., Lo M.C., Lin K.C., Pan C.C., Chang R.Y. 1990. Characterization of filamentous bacteriophage phi Lf from Xanthomonas campestris pv. campestris. J Gen Virol. 71 :1881-4. Wang T.W., Tseng Y.H. 1992. Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage phi Lf. Lett Appl Microbiol. 14:65-8. Yang, B. Y., and Y. H. Tseng. 1988. Production of exopolysaccharide and levels of protease activity in pathogenic and non-pathogenic strains of Xamthomonas campestris pv. campestris. Bot. Bull. Academia Sinica. 29:93-99. Zasloff M. 2002. Antimicrobial peptides of multicellular organisms. Nature. 415:389-95.
摘要: 
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that causes black rot in crucifers. Many strains of Xcc are β-lactam resistant via expressing β-lactamase (AmpC/Bla) constitutively. Previous studies showed that transcription from bla promoter is regulated by a global transcriptional regulator, Clp (cAMP receptor protein-like protein). Mutation in Clp changes constitutive expression of bla to an ampicillin-inducible manner. This study aimed to explore the possibility in application of Ap-inducibility in the clp-deficient mutant strain, TC817, derived from Xc17. For the testing, several plasmids were constructed, which carried genes encoding lysis protein from the Xcc phage phiL7 or antimicrobial peptide (AMPs) under control of the bla promoter. These plasmids were introduced into TC817, and the resultant transformants were incubated in ampicillin (Ap)-containing medium. Results showed that growtn rate of the cells was reduced, suggesting that Ap can induce bla promoter to express lysis protein and AMPs. Thus cell growth was inhibited by Ap. Growth inhibition was also observed with P20H, a mutant derived from Xc11A. The results also indicated that lysis protein p27 (hollin) can effectively inhibit the growth of P20H. In the second part of this thesis, the filamentous phage phiLf of Xcc was used to construct a new vector system for expression of p27 gene driven by the plant-inducible promoters (PIP) of hrpB, hrpE and hrpF. The recombinant RF DNA were then introduced into Xcc. However, no lysis was observed for the recombinant phiLf-infected P20H in XVM2 medium in which PIP would activate the PIP promoters. Then we replay PIP by bla promter and incubate cell on Ap-containing medium. Results showed that cell growth was inhibited after recombinant phage infection.

大多數的十字花科蔬菜黑腐病菌 Xanthomonas campestris pv. campestris (Xcc) 皆能產生β-lactamase (AmpC),因此,普遍具有抗β-lactam 類抗生素 (例如:penicillins、cephalosporins 等) 的特性。本實驗室過去研究顯示 Xcc strain 17 (Xc17) 的多元調控轉錄因子Clp (cAMP receptor protein-like protein) 基因突變後,β-lactamase 表現由持續型轉變為受誘導型。本實驗利用 Xc17 clp 突變株 TC817 的 bla 啟動子受 ampicillin (Ap) 誘導表現的特性探討其應用性。首先,測試 φL7溶裂蛋白對菌體生長的影響,在 TC817 中 p27-29.1 基因產物抑菌效果較明顯,而p27 (holin) 抑菌效果不佳;但 p27 對 Xc11A突變株 P20H 則有明顯抑制菌體生長的效應。進一步將具有殺菌功能的溶裂蛋白 (p27) 或抗菌胜肽 lactoferricin (LF) 之 DNA 序列,選殖於 arabinose (ara) 或 bla 啟動子下游,再將構築完成之質體轉殖送入 TC817 及 P20H。結果顯示在含有誘導物 arabinose 或抗生素 Ap的 plate中,TC817 與 P20H 轉殖株之生長受到抑制,存活率下降,表示相似於 ara promoter 受到 arabinose 誘導表現,bla promoter 可受到 Ap 誘導表現毒殺蛋白及抗菌胜肽,導致細菌的生長受到影響。然而,此誘導表現抗菌胜肽抑制細菌生長的效應,在固體培養基上較明顯,在液體培養基中則否。前人發現 P20H 可被線狀噬菌體 φLf 感染,且感染率較高,適合作為宿主菌株。本實驗中以線狀噬菌體 φLf 為基礎,發展載體系統,將具有毒殺菌體能力的 p27 基因,構築於具有 plant-inducible promoter (PIP) 特性的 hrpB、hrpE、hrpF 啟動子下游。將線狀重組噬菌體感染宿主後,在仿植物環境的貧乏培養基 XVM2中,細菌的生長曲線及存活率並沒有明顯下降現象。另以線狀噬菌體 φLf 為基礎,發展載體系統,將具有毒殺菌體能力的 p27基因,構築於具有bla啟動子下游。測試重組噬菌體感染宿主後,在含有Ap的固體培養基,菌體的溶裂情形較明顯,利於進一步應用在生物防治上。
URI: http://hdl.handle.net/11455/92234
Rights: 同意授權瀏覽/列印電子全文服務,2015-08-28起公開。
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