【作者】 张静；

【导师】 张明；

【作者基本信息】 安徽农业大学 ， 微生物学， 2006， 硕士

【摘要】 Tat蛋白转运系统是大肠杆菌中重要的蛋白质转运系统,在该系统中蛋白质的转运受到多种因素的影响。本研究的目的是建立研究转运效率的技术方法,并尝试通过该方法筛选转运效率突变株,为进一步的研究提供实验材料。研究表明利用细菌素作为报告蛋白,在液体和固体培养中细菌的生长密度可反映Tat转运系统转运效率的高低,因而可作为检测转运效率突变菌株的手段。比较了菌株在液态摇瓶和固体平板培养的生长密度,结果表明液体发酵培养所测得的生长曲线和固体平板点种培养后生长的菌苔浓密度均能通过杀伤作用反映菌株的转运效率。为了便于筛选,选用固体平板对诱变后菌株进行初步筛选,进一步利用液体培养加以验证。比较RR(twin-Arg translocation)和KK(twin-Lys translocation)两种信号肽对细菌素转运的能力,发现RR信号肽对细菌素的高转运率使对自身细胞有强烈的杀伤作用,造成后续检测结果工作的困难,因而选用KK信号肽用于对突变菌株的筛选。本研究通过离子束作为诱变剂对大肠杆菌MC4100A进行诱变,并导入编码有Tat信号肽和细菌素基因的质粒,作为转运自杀式探针检测系统,筛选大肠杆菌Tat转运系统转运效率有变化的突变菌株。进一步分析初筛后细胞排列特性,在M9培养基中的生长性能和在SDS中培养特征和属性鉴定,从而确认Tat蛋白质转运系统转运效率可能产生变化的菌株。通过以上方法筛选出了10株在阿拉伯糖培养中生长密度有变化的菌株。进一步分析10株菌在M9培养基中和含有2%SDS平板上的生长状况,以及菌株形态及生理生化特征,初步确定了2株转运效率有变化的菌株。更多还原

【Abstract】 The Tat translocation system plays important roles in Escherichia coli. The efficient translocation can be affected by multifold factors. In this research the method is afforded to separate the mutants of different efficient translocation that could be studied further more. The results revealed that the efficient translocation of ColV reporter protein could be showed by the growth of strains in liquid medium and on solid plate.Comparison the growth of various strains cultivated in liquid medium with solid medium, the result displayed that both of methods could show the efficient translocation. In order to do easy, strains were inoculated in solid medium firstly, and then were cultured in the liquid medium.Comparison the wild-type strain carrying RR-ColV(twin-Arg translocation) with KK-ColV (twin-Lys translocation) of the efficient translocation for ColV, the result showed that the strain carrying plasmid of RR-ColV could be killed severely. So the KK signal peptide was chosen in the experiment.The plasmid containing the genes of the colicinV and the Tat signal peptide was transfered into MC4100A, one of wild type strain of Escherichia coli, directly by the low energy nitrogen ion implantation. The plasmid was just used as system of the translocation-suicide probe to select out the mutants of different efficient translocation via Tat system. Moreover, the analysis for the characteristic array of the mutant cells, the growth in the medium of M9, the susceptibility to SDS, morphology characteristic and some biochemical characteristic were adopted further to identify the mutants that the efficiency were different.Ten mutants having different efficient translocation for ColV were singled out by above methods. Then they were cultured in the medium of M9 and the medium added 2% SDS respectively. At last two mutants were confirmed that the efficient to exporting were different to MC4100A×KK-ColV.更多还原