Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21245
標題: 十字花科蔬菜黑腐病菌beta-lactamase調控基因之功能與特性分析
Characterization and Functional Analysis of beta-lactamase Regulatory Gene from Xanthomonas campestris pv. campestris
作者: 陳義元
Chen, Yih-Yuan
關鍵字: 抗生素調控
beta-lactamase
報導基因
ampR
出版社: 分子生物學研究所
摘要: Xc11的bla及ampR基因皆已選殖並定序完成。分析序列發現其轉錄方向相反,兩基因之轉譯起始點相隔100 bp,且預測之啟動子區域重疊。但目前有關其Xc11 *-lactamase的表現調控機制尚有許多不清楚的地方。因此,為了進一步瞭解AmpR所扮演的角色,首先利用同質性重組的突變方式使Xc11染色體上的ampR基因失去功能,藉以得到Xc11 ampR基因突變株,命名為Xc11RM。經由抗生素感受性實驗、最小抑制濃度實驗及*-lactamase酵素活性分析,發現當菌體內缺乏AmpR時,Xc11*-lactamase活性及對抗抗生素的能力下降,顯示 AmpR在調控Xc11 *-lactamase的表現上,扮演一正向調節子的角色,且*-lactamase之表現為AmpR-dependent。此外,亦發現*-lactamase酵素活性表現型態為持續性的( constitutive )表現。互補株與野生株之*-lactamase酵素活性表現,前者為後者的3-4倍,推測AmpR量的增加,也會影響造成酵素活性相對增加。前人曾以Xc11之ampR基因為探針進行Northern hybridization,結果只偵測到相當微弱之訊號。本實驗中以AmpR抗體進行Western blotting分析,亦無法在Xc11中偵測到訊號。顯示ampR基因之RNA半衰期可能極短,且表現量相當少。本研究中,以luxAB為報導基因,將bla啟動子分別轉殖於啟動子測試質體pFY7及pFY7-Gm的報導基因上游,構築成質體pBX及pBX-Gm。而比較Xc11 ( pBX )及Xc11RM ( pBX-Gm )之螢光表現值,推測AmpR可能有輔助RNA polymerase轉錄bla基因起動之功能。另外,將bla-ampR基因轉殖至泛寄主載體pFY13-9上,將構築完成之質體轉殖送入一Aps之X. campestris之病原小種,Xcm38中。分析結果顯示bla-ampR系統在Xcm38無法作用,推究其原因可能為缺乏類似signal transducer,例如:AmpG等因子之故。
In the previous work, a novel b-lactamase (bla) and its regulatory gene, ampR, has been characterized in the gram-negative phytopathogenic bacteria Xanthomonas campestris pv. campestris 11 (Xc11). The ampR gene was located directly upstream from bla and the promoters of ampR and bla overlapped and divergently directed RNA transcription. In this study, an ampR mutant, Xc11RM, was constructed by the technique of gene replacement and used to investigate the role of ampR in Xc11. The results of antibiotic sensitivity test, minimal inhibitory concentration ( MIC ) test, and b-lactamase enzyme activities assays indicated that AmpR acts as a positive regulator for the expression of b-lactamase. Addition of b-lactam antibiotic to growing culture did not induce the expression of bla, indicating that the b-lactamase is constitutively expressed in Xc11. The b-lactamase activity of Xc11RM (pFJ-ampR) is 3- to 4- fold higher than that of Xc11, suggesting that the amount of AmpR in Xc11 can influence the expression of b-lactamase. According to our results, it is proposed that AmpR may regulate the transcription of bla by bending DNA for a better recognition between RNA polymerase and bla promoter.
URI: http://hdl.handle.net/11455/21245
Appears in Collections:分子生物學研究所

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