【作者】 李美菊；

【导师】 沈萍； 陈向东；

【作者基本信息】 武汉大学 ， 遗传学， 2005， 博士

【摘要】 本论文对地衣芽孢杆菌碱性蛋白酶基因进行了克隆,并转入产蛋白酶缺陷的枯草芽孢杆菌DB104菌株中,从而构建产蛋白酶的遗传工程菌株(GeneticallyEngineered Strains)。采用荧光定量PCR、核酸分子“光开关”、遗传转化等方法对该重组菌株释放DNA的情况进行了研究,并通过对两个突变株(BD1704和MD301)释放DNA情况的研究,初步探讨了枯草芽孢杆菌释放DNA的机制。获得了如下研究结果: 1.以大肠杆菌—枯草芽孢杆菌穿梭质粒pBE2为载体,将包含不同调控区域的地衣芽孢杆菌L106碱性蛋白酶基因片段分别克隆到枯草芽孢杆菌和大肠杆菌中。采用生物学和微量热技术对各重组菌株的研究结果证明,该基因的3′非编码区和5′上游序列均对该基因在枯草芽孢杆菌中的表达具有重要意义,只有携带该蛋白酶基因全序列的106-1片段(pAPR8*1)能在产蛋白酶缺陷的枯草芽孢杆菌DB104菌株中进行转录并获得成功表达。此外,上述基因克隆片段最初在大肠杆菌中均未得到表达,而各重组菌株间生长代谢产热曲线的微量热法分析结果显示,该基因没有得到成功表达的原因可能是其上游序列中的启动子功能不够强,通过更换含有强启动了的载体,使该基因最终在大肠杆菌中获得了表达,这说明微量热技术在某些情况下有可能为分子生物学操作提供辅助指导。更多还原

【Abstract】 The work of this dissertation was aimed to constructing an engineered strainDB104/pPAR8-l producing protease by cloning the alkaline protease gene (aprE) fromBacillus licheniformis L106 into Bacillus subtilis strain DB104 which was a deficiencyof protease producing and studying DNA releasing of this genetically engineered strainusing real-time fluorescence quantitative PCR, nucleic acid molecular "light switch"compound Ru(phen)2(dppz)~2+ and transformation methods. We discussed the preliminarymechanism of DNA releasing from B.subtilis through studying on two mutation strains(B. subtilis BD1704 and MD301). The results achieved were listed below:1. By employing the Escherichia coli and Bacillus subtilis shuttle plasmid pBE2,several alkaline protease gene fragments of L106 with different regulation regionswere cloned in E.coli and B.subtilis respectively. These recombinant strains werestudied using biological and microcalorimetric technique, the result showed that the3’non-coding region of the gene was as important as that of the 5’upstream region forits normal express in Bacillus. Only 106-1 DNA fragment containing the wholesequence of alkaline protease gene (aprE) was transcribed and successfully expressedin B.subtilis DB104 strain. In addition. aprE gene failed to express in the strain ofE.coli at first. However, the metabolizable heat-out of several recombinant strainswere detected using the microcalorimetric technique. It was found that there wereobvious differences among their metabolizable heat-out curves. The result of analysisrevealed that the reason of failing to be expressed in E.coli may be the weakerpromoter in the upstream of this gene. So two aprE gene fragments (106-3 and 106-4) were respectively sub-cloned in another plasmid vector containing a stronger promoter and then this gene was successfully expressed in E.coli. All of these proved that microcalorimetric technique might give some suggestions for genetic engineering operation under some conditions.2. A new faster and more sensitive experiment method was used for the first time to study the rule of aprE gene released by B. subtilis recombinant strain C ( DB104/pAPR8-l). The result suggested that there was certainly a peak of this gene secreted in early exponential growth phase and the maximal concentration of this gene was 1.5×10~12 copies per μl. And in the post stationary phase a large number of this DNA fragment was obtained in the culture of strain C. Furthermore the transformation activity of plasmid pAPR8-l released by strain C was detected using the transformation assay. It was revealed that the plasmid pAPR8-l secreted in early exponential phase had transformation activity. Moreover there was the precise time correlation between the appearance of active release of DNA and the achievement of competence. On the other hand, the enzyme activity of the expressed product of aprE gene (alkaline protease) was detected. The result revealed that there were activated alkaline protease in the culture during the releasing aprE gene in the early exponential phase. So we presumed that there was precise time correlation between the secretion of DNA and its product (alkaline protease).3. The activity of DNase released from this recombinant strain was detected during the whole bacteria growth period. The result indicated that the activity of DNase existing in the cell-free filtrate was very high in the early exponential growth phase (ahead of the time of DNA secretion). It was presumed that DNase in this stage was excreted by this strain. Furthermore after this stage, the activity of DNase decreased. Presuming that DNase could be adsorbed to the cell surface or combine with some kind of components to form compounds. So the conformation of DNase was changed and the chance of contacting with DNA was reduced.4. A more sensitive and specific experimental method was firstly employed to detect the whole quantity of DNA released from B. subtilis recombinant strain C using nucleic acid molecular "light switch" Ru(phen)2(dppz)2+ as fluorescent probe. The result indicated that the whole quantity of DNA obviously increased and formed a peak inearly exponential growth phase. Based on the rule of bacteria growth, cell lysis could not happen in this phase (also proved by the result of microscope assay). So this result verified the conclusion made using fluorescent quantitative PCR. 5. A complex regulation network of the physiological metabolism exists in B. subtilis which concerned lots of indispensable genes and related regulation genes. mecB and degU were the key regulation gene in this network. The DNA in culture released from two mutant strains BD1704 (mecB31) and MD301 (degU146) were detected by fluorescence quantitative PCR. The results indicated that it was different dramatically between the rule of DNA released from these two mutant strains and that of wild type strain. It was presumed that mecB and degU maybe involve in the regulation of some way related to the DNA release. And the DNA release and the development of competence maybe share some regulation factors in part of regulation way. We further presumed that DNA release system of B. subtilis might have a complex information regulation network as well as competence development, sporulation, exoenzyme synthesis and secretion, and cross with regulation system of competence development, sharing some regulating way or regulation factors.更多还原