【作者】 应南娇；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2008， 博士

【摘要】 辐射抗性微生物是一种重要的极端微生物资源,其对环境的适应性生长是微生物与外界环境相互作用的结果。耐辐射球菌(Deinococcus radiodurans)是目前所知的最抗辐射的生物之一,具有超强的辐射抗性和完善的DNA修复机制。关于辐射抗性微生物的起源与进化,人们虽然提出了各种假说,但都缺乏相应的实验证据,其起源与进化机制至今仍然是一个谜。本文研究主要以非辐射抗性菌株大肠杆菌(Escherichia coli)K-12为材料,通过83次γ射线辐射进化诱导,筛选得到具有一定辐射抗性能力的突变菌株,然后通过基因组序列比较、差异蛋白质组、部分基因的转录水平和蛋白质氧化损伤等方面的分析,研究大肠杆菌在电离辐射压力下的适应性进化机制。同时分析了耐辐射球菌(D.radiodurans)在γ射线辐射前后的细胞内外蛋白质组表达水平差异,探讨耐辐射球菌超强的辐射抗性与DNA损伤修复机理。1.首先以大肠杆菌(E.coli)K-12为起始菌株,在反复多次的γ射线电离辐射后筛选得到具有一定辐射抗性能力的细菌。对菌株IR58、IR21和IR51进行逆境生存率分析,结果表明IR58、IR21和IR51对γ射线辐射抗性能力增强,同时IR58和IR21对紫外线UV和丝裂霉素C也产生了一定的抗性。2.选取具有较强辐射抗性能力的菌株IR58和IR21,通过Solexa GenomeAnazyler系统进行基因组测序。将IR58和IR21的基因组序列与大肠杆菌K-12的标准基因组序列比较后发现,IR58和IR21基因组在辐射损伤的适应性进化过程中经历了大量的基因突变与DNA片段复制事件。在IR58和IR21中找到了正在进行的基因复制现象,这些基因在核苷酸水平上有很高的相似度,同时也找到了经过插入突变与删除等的“去功能化”作用后分化较厉害的序列。在IR58和IR21中有大量分散存在于基因组的SNP位点,来源于受外界环境损伤后基因序列内的单碱基置换。进一步分析发现在IR58菌株中,uvrB,mutM、lexA、hepA、danX等5个DNA修复和复制相关基因发生了碱基替换和插入删除突变,而在IR21菌株中,recC、recJ,dnaE,mutM、hepA和mutS等6个基因也发生了碱基替换和插入删除等突变。基因组比对的结果表明,IR58和IR21两个菌株发生了大量的点突变、基因分子复制重排和DNA片段的缺失等事件。3.利用二维液相色谱分离系统结合质谱鉴定方法分析了大肠杆菌K-12和IR58、IR21在γ射线诱导下蛋白质组表达水平差异。与K-12相比,有144个蛋白质在IR58和IR21中表达水平升高或降低的变化趋势是一致的,通过质谱分析后成功鉴定了39个蛋白。而有38个蛋白的变化趋势是不同的,通过质谱分析后成功鉴定15个蛋白。鉴定得到的这些蛋白归属于不同的生物学功能,其中有4个变化趋势相同的蛋白曾被报道与DNA修复机制相关。4.分析了辐射抗性菌株IR58和IR21中部分DNA修复相关基因的转录水平,发现参与切除修复途径的基因特别是uvrABC基因的转录水平都有不同程度的提高,参与同源重组途径的recA,ssb,recBCD和recFOR等相关基因的转录水平也有提高。同时使用DNPH法分析了细胞内蛋白质的氧化损伤程度,发现IR58和IR21在受到γ射线损伤后,氧化损伤的程度比野生株K-12要低,说明IR58和IR21细胞内保护蛋白质免受氧化损伤的能力提高了。5.为了研究耐辐射球菌的辐射抗性和DNA修复相关机制,应用蛋白质组学的分离和鉴定方法考察了耐辐射球菌胞内蛋白和胞外分泌蛋白在γ射线辐照诱导下表达水平差异。其中,胞内有26个蛋白质的表达水平有明显的增加,胞外有29个蛋白质发生了明显的表达水平变化。通过生物质谱鉴定得到部分蛋白质的信息,发现这些蛋白质在细胞中分别归属于不同的生物学功能,而且大部分蛋白质未曾报道与辐射抗性机制相关。对细胞内总蛋白表达谱分析发现有两个蛋白(SSB、PprA)曾报道与DNA修复相关,而有5个蛋白的功能是参与细胞的调控与信号转导。对胞外蛋白的鉴定与分析发现,有1个蛋白曾报道与DNA修复相关,有8个蛋白与细胞物质的转运和分泌相关。更多还原

【Abstract】 Extremophiles have a number of potential applications,which would benefit from the use of radiation-resistant microorganisms.It begins from the flexibility of microorganisms to adapt to a wide spectrum of extreme environments.Deinococcus radiodurans is one of the most radiation-resistant organisms described to date.Studies show that the resistance is due to D.radiodurans’s extremely proficient and accurate DNA repair process.Evolutionary hypothesis about the origin of radiation-resistant microorganisms seems to show a lack of experimental evidence.The origin and evolution of radiation-resistant microorganisms is still remaining unknown in large part.In this study,the founder strain was non-radiation resistant wild-type Escherichia coli K-12.Wild-type E.coli K-12 was exposed to 83 successive rounds ofγ-ray radiation,allowing for full recovery in between each exposure.Strains IR58 and IR21 selected from the final round of irradiated culture showed a dramatic increase in radiation resistance compared to its parental wild-type E.coli strain.In order to understand evolution mechanism during adaptation to irradiation,we applied the whole genome resequencing,comparative proteomics,gene transcription levels and protein oxidation analysis of IR58 and IR21.In order to reveal the mechanisms of extreme radioresistance and DNA repair in D.radiodurans,we also examined intracellular and extracellular proteome changes in D.radiodurans followingγ-irradiation.1.E.coli K12 was forced through cycles of rapid evolution by iterative exposures to doses ofγ-irradiation for 83 cycles.Three robust strains selected from the final round of irradiated culture were named IR58,IR21 and IR51.The three strains showed a dramatic increase in radiation resistance compared to its parental wild-type E.coli strain.IR58 and IR21 also showed a moderately resistant to UV and more resistant to mitomycin C.2.The whole genome of IR58 and IR21 were resequenced by the high-throughput Solexa Genome Anazylers sequencing system.Our comparative study revealed that IR58 and IR21 underwent numerous events including genetic mutation,gene duplication rearrangement and segment deletion during irradiation adaptive evolution.We found differentiation sequences had undergone many non-functionalization events in both IR58 and IR21,and also gene duplication. Numerous single nucleotide polymorphisms(SNPs)were dispersedly exist in whole genome of both IR58 and IR21.We also found that five genes(uvrB.mutM.lexA. hepA and danX)in IR58 and six genes(recC.recJ.dnaE.mutM.hepA and mutS)in IR21 occurred base substitution,insetion or deletion.Based on comparative genomic data,we propose that the whole genome of both IR58 and IR21 suffered numerous events during irradiation adaptive evolution,such as point mutations,gene duplication rearrangement and segment deletion.3.We developed the ProteomeLab PF-2D fractionation system to carry out large scales protein expression analysis of cell lysates from IR58,IR21 and K-12. Compared with wild-type K-12,expression level of 144 proteins had significantly changed in both IR58 and IR21.But 38 proteins were changed in different trends in IR58 and IR21.39 of 144 proteins and 15 of 38 proteins were identified using matrix-assisted laser desorption ionisation time of flight mass spectrometry after tryptic digestion.These proteins exhibited various cellular functions,and four of them were reported to relate to DNA repair.4.The transcription levels of genes related to DNA repair and replication were analyzed in IR58 and IR21.Compared with E.coli K-12,transcription levels of many important genes in IR58 and IR21 were increased,such as uvrABC,recA,ssb,recBCD and recFOR.Protein oxidative injury was analyzed using the 2,4-dinitrophenylhydrazine(DNPH)procedure.Afterγ-irradiation,IR58 and IR21 had lower protein carbonyl content compared with E.coli K-12.The results indicated that the ability to protect proteins in IR58 and IR21 might be well developed,thus becoming a survival factor with irradiation.5.In order to reveal the mechanisms of extreme radioresistance and DNA repair in D.radiodurans,we examined intracellular and extracellular proteome changes following T-irradiation using 2-D PAGE and silver staining.Among the identified proteins,most of them have not previously been reported to be relevant to radioresistance.Five proteins in intracellular proteome were classified in signal transduction and regulation,and two proteins(SSB,PprA)were reported to have concern to DNA repair.Seven in extracellular proteome are classified in transport and metabolism,and one is involved in intracellular trafficking and secretion.更多还原