【作者】 周正富；

【导师】 林敏；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 耐辐射异常球菌(Deinococcus radiodurans)具有极强氧化胁迫抗性和电离辐射抗性。该菌中发现的IrrE是异常球菌属特异的全局调控因子,在DNA损伤修复、胁迫应答以及保护途径起到一个中心调控作用,并能显著增强模式生物大肠杆菌和烟草的耐盐能力。然而,IrrE在异源生物中的作用机制尚不清楚。本论文将IrrE导入大肠杆菌中,研究其对受体细胞氧化、高温和盐胁迫抗性的影响,并进行了蛋白质组和转录组分析。取得主要研究成果如下:1.在正常生长条件下,IrrE蛋白能够延长大肠杆菌生长对数期,提高细胞生物量,表明IrrE能增强大肠杆菌一般胁迫抗性。BioLog 95底物利用分析表明,与对照菌株相比,重组菌株对22种碳源的利用能力显著降低,相反对山梨醇的利用能力显著提高。蛋白质组和转录组分析结果显示,IrrE重组大肠杆菌中参与海藻糖的合成、核苷酸合成、碳源利用、氨基酸利用、酸抗性等途径的基因表达差异显著。一系列具有调节功能的基因表达差异显著,其中evgA、appY、gadE、gadW、gadX、yhiF和asnC上调,purR、betI、cynR、mhpR、prpR、tdcA和kdgR下调。表明异源表达的IrrE可能通过转录级联放大反应在宿主细胞中发挥全局调控的作用。2.不同盐浓度冲击1 h后,IrrE能够提高大肠杆菌的存活能力;在1.0 M盐浓度下培养24 h,IrrE能提高大肠杆菌的生长能力;在高温和过氧化氢处理条件下,IrrE能提高大肠杆菌的存活能力。上述结果表明,IrrE在大肠杆菌中异源表达增强其胁迫抗性。为了研究IrrE提高大肠杆菌耐盐抗性的全局调控机制,本论文分析了1.0 M盐冲击下重组大肠杆菌与对照菌的蛋白图谱。结果表明,高浓度盐冲击下,重组菌株中126个蛋白表达显著上调,110个蛋白表达显著下调。其中包括一系列环境胁迫诱导蛋白,如胁迫反应转录因子RpoS、胁迫反应调控蛋白Dps、分子伴侣DnaK、热激蛋白HslU、渗透胁迫诱导蛋白OsmY、噬菌体诱导蛋白PspA、过氧化氢酶KatE、一般胁迫反应蛋白YhbO、分子伴侣Tig和胁迫条件诱导蛋白酶Lon等表达量显著提高。此外,盐冲击条件下细胞内甘油降解途径相关酶蛋白表达下调,同时甘油含量测定结果表明,重组菌株胞内甘油含量明显提高。表明IrrE引起胞内甘油积累,是重组大肠杆菌增强盐胁迫抗性的策略之一。3.迄今发现的4个IrrE均来自异常球菌属,其蛋白一级结构相对保守。N-端结构域的氨基酸序列有较大差异,但均含有一个锌依赖型多肽酶结构。为研究N-端结构域的生物学功能,对耐辐射异常球菌IrrE的N-端进行了截短分析,回补耐辐射异常球菌IrrE突变株发现,N-端的18、26、43个氨基酸残基缺失的IrrE突变体能恢复突变菌株的紫外和电离辐射抗性,但N-端的160个氨基酸残基缺失不能恢复其抗性。本研究将为揭示微生物的环境适应进化和盐胁迫抗性机制,并进一步开展IrrE基因改良植物与微生物性状等研究奠定工作基础。更多还原

【Abstract】 Deinococcus radiodurans is unparalleled among all life in its capacity to survive oxidative stresses, and ionizing and ultraviolet (UV) radiations. IrrE is a global regulator, without ortholog in other genera, that greatly enhances the DNA repair capability and the radiation resistance of D. radiodurans. Our study demonstrated that heterologous expression of IrrE confers significantly enhanced salt tolerance in both E. coli and tobacco. However, the mechanisms and regulation of the enhanced stress resistance of IrrE-expressing cell is not fully understood. Furthermore, the heterologous expression of irrE increased tolerance to oxidative stress, heat shock and other stresses in E. coli. To better understand the global regulatory effects of IrrE we carried out a combined transcriptome and proteome analysis comparing of E. coli expressing irrE under normal and salt (NaCl) stress conditions.The main results obtained are as follows:1. In this work, we investigate that the IrrE-expressing strain was displayed better growth than the control strain with higher maximal cell density in LB medium. The carbon source utilization profile of E. coli expressing IrrE was determined using Biolog. IrrE greatly affected the types of substrates that were used and the efficiency with they were used. To better understand the global effects of IrrE on the regulatory networks, we carried out combined transcriptome and proteome analysis of E. coli expressing the IrrE protein. Our analysis showed that a large number of host genes with a significant change in expression, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon source utilization, amino acid utilization, acid resistance, a hydrogenase and an oxidase. Also regulated were the EvgSA two-component system, the GadE, GadX and PurR master regulators, and 10 transcription factors (AppY, GadW, YhiF, AsnC, BetI, CynR, MhpR, PrpR, TdcA and KdgR). These results demonstrated that IrrE acts as global regulator and consequently improves abiotic stress tolerances in the heterologous host E. coli.2. We investigate that IrrE can be utilized to improve tolerance to various abiotic stresses. The results demonstrated that the heterologous expression of IrrE in E. coli increased tolerance to transient and continuous salt stress and heat shock, in addition to oxidative stress resistance. To investigate the regulatory mechanism of IrrE in response to salt stress, we performed comparative proteomics analyses on the IrrE-expressing strain and the control strain under salt stress condition. We detected significant changes for 124 proteins, with 66 proteins being upregulated in the IrrE-expressing strain. These up- or downregulated proteins can be grouped into 13 classes based on their common functional characteristics. Among them, a set of stress responsive proteins were upregulated by IrrE, including the molecular chaperone DnaK, the heat shock protein HslU, the osmotically inducible protein OsmY, catalase HPII KatE, general stress protein YhbO, the trigger factor Tig, the stress-inducible ATP-dependent protease Lon and stress response protein Dps. We found significant induction of general stress regulator RpoS, which exhibits a 3-fold-higher expression in the IrrE-expressing strain. After NaCl shock, the glycerol-degrading enzymes were down-regulated. The glycerol level in IrrE-expressing cells reached 37 nmol/mg dry weight and was approximately 2-fold higher than that in control cells.3. IrrE homologs from different Deinococcus species showed considerable N-terminal variation. Guided by the sequence alignments, four irrE deletions which removed 18, 26, 43 and 160 aa from the N-terminus of the 328 aa protein. Only the removal of 160 aa affected the UV and ionizing radiation resistance, abolishing it completely.更多还原