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【目的】研究铜绿假单胞菌弹性蛋白水解能力相关基因。【方法】应用人工Mu转座技术构建铜绿假单胞菌野生型菌株PA68的转座突变文库,从2000多个突变子中筛选得到4株弹性蛋白水解能力改变的突变子,并通过克隆及测序获得转座子插入位点侧翼的序列。将铜绿假单胞菌弹性蛋白酶结构基因lasB的转录启始区序列整合入载体pDN19lacΩ并将该重组质粒电转化入野生型菌株PA68及4个突变株中,对报告基因在不同菌株中的表达水平进行测定。【结果】发现4个突变株中Mu转座子分别插入lasA、galU、xcpZ和ptsP 4个基因。ptsP基因失活的突变株中,lasB基因的转录水平是野生型菌株的7%,xcpZ和lasA基因的失活使lasB基因的转录水平分别降低为野生株的54%和75%,galU基因的插入失活使lasB基因的转录上升了1倍。【结论】推测ptsP和galU基因很可能直接或间接地调控着弹性蛋白酶的生物合成。
【Objective】 The objective of this study was to investigate the genes involved in the elastase proteolytic activity of Pseudomonas aeruginosa. 【Method】 The transposable library of Pseudomonas aeruginosa wild-type strain PA68 was constructed by artificial Mu transposon technology. Four mutants with altered elastolytic ability were screened from more than 2000 mutants and cloned and sequenced The sequence flanking the transposon insertion site was obtained. The transcription initiation region of lasB gene of Pseudomonas aeruginosa elastase structural gene was integrated into pDN19lacΩ vector and electroporated into wild-type strain PA68 and four mutant strains. The expression level of reporter gene in different strains Measurement was performed. 【Result】 Mu transposon was found to insert four genes of lasA, galU, xcpZ and ptsP respectively in four mutant strains. In the mutant with ptsP inactivation, the transcription level of lasB gene was 7% of the wild-type strain. The inactivation of xcpZ and lasA genes reduced the transcription level of lasB gene to 54% and 75% Insertion inactivation doubled the transcription of the lasB gene. 【Conclusion】 It is speculated that ptsP and galU genes may directly or indirectly regulate elastase biosynthesis.