【作者】 刘阳；

【导师】 张松林； 章高森；

【作者基本信息】 西北师范大学 ， 工程硕士（专业学位）， 2020， 硕士

【摘要】 沙漠环境由于其极端干旱、干燥、强烈紫外线辐射（UV-A和UV-B波段紫外射线）、温差变化大和降水极少的恶劣特征,导致大部分动植物在沙漠中无法生存。然而,在沙漠的生态系统中,微生物作为为数不多的生物类群,却能够顽强生存。在沙漠生态系统中,微生物作为该生态系统的主导生物,能够顽强应对沙漠中高辐射、高度氧化、极度干旱而生存;这就说明沙漠微生物在这样的环境形成了抗逆性机制来保护自生的生存与传代。通过对沙漠中可培养微生物的抗辐射抗氧化机制研究,可以为辐射损伤和氧化损伤的抗性修复资源提供科学依据和理论支持。本文首先通过对塔克拉玛干沙漠中的微生物培养,对其中的可培养微生物的多样性进行研究;再通过UV-C和γ射线辐照的手段,对可培养细菌进行了抗辐射筛选。最后,对筛选出的强抗辐射菌株和中国科学院甘肃省极端环境微生物资源与工程重点实验室提供的库木塔格沙漠的抗辐射菌株进行抗氧化研究和TKL1057菌株的代谢组分析。研究结果表明:1.从塔克拉玛干沙漠中共分离出2216株可培养细菌;分属于不同的4个门,分别是放线菌门（Actinobacteria）、厚壁菌门（Firmicutes）、拟杆菌门（Bacteroidetes）和变形菌门（Proteobacteria）;43个不同的属;352个不同的种。在门水平上,首次发现塔克拉玛干沙漠可培养微生物中,在相对丰度上放线菌门（Actinobacteria）最高;从CFU来看,放线菌门（Actinobacteria）和厚壁菌门（Firmicutes）均是优势的门。2.在塔克拉玛干沙漠中从北向南采集的9各个样点中,不同种微生物的分布生物多样性不同。在门水平上,TOC、TC、TN、WC、EC含量和pH值与线菌门（Actinobacteria）、厚壁菌门（Firmicutes）和变形菌门（Proteobacteria）的细菌丰度之间呈现显著的相关关系。不同的环境因子对不同门的细菌的分布多样性和种类多样性都具有显著相关影响,且这种影响的生态响应强弱不同3.对塔克拉玛干沙漠样品培养分离出的细菌进行UV-C和γ射线辐照,筛选出了抗辐射能力强的细菌;且除了拟杆菌门没有筛选出比Deinococcus radiodurans抗辐射能力强的细菌外,其他三个门共有10株细菌在HR（6000 J/m² UV-C）的辐照条件下抗辐射能力比阳性对照强。研究发现:TKL1057（Kocuria indica）、TKL798（Streptomyces griseoviridis）、TKL860（Citricoccus zhacaiensis）、TKL1152（Planococcus citreus）、TKL19（Gracilibacillus thailandensis）、TKL588（Bacillus subtilis subsp.Inaquosorum）、TKL606（Pseudomonas xanthomarina）、TKL865（Paracoccus hibisci）、TKL855（Pseudochrobactrum saccharolyticum）和TKL35（Altererythrobacter soli）。4.TKL1057（Kocuria indica）和TKL1152（Planococcus citreus）这两株菌的抗γ辐照能力强于Deinococcus radiodurans,而γ射线辐照下其它菌株的存活率几乎接近于0,说明TKL 1057和TKL 1152抗辐射能力较强的菌株。5.通过抗氧化筛选发现:不同抗辐射菌株的抗氧化存活率为TKL 1057>K81>TKL 1152>TKL 798>AQ（Deinococcus radiodurans）>K146>K202>TKL 865>K59>BL（阴性对照）;同时还发现不同抗氧化菌株的抗性胁迫浓度不同。抗氧化菌株在氧化胁迫下对DPPH、OH2和O^2-2自由基都具有良好的清除能力,且MDA含量在氧化胁迫下也较高。6.对TKL1057（Kocuria indica）进行辐射和氧化胁迫后测定其代谢组,发现:经过辐射和氧化胁迫后,TKL1057的代谢产物和代谢通路确实发生显著性改变;辐射后有61种代谢产物进行了表达,氧化后有40种代谢产物进行了表达,辐射和氧化处理后表达的代谢产物种有40种是相同的,同时不同处理下代谢差异物的分析说明辐射后的细胞代谢主要以糖类基础代谢为主合成次级代谢修复产物,氧化后的代谢主要以有机酸代谢为主进行次级代谢修复产物的合成。更多还原

【Abstract】 Because of its extreme drought,dryness,high ultraviolet radiation(a large number of UV-A and UV-B ultraviolet rays),large temperature difference and a little precipitation,most of the animals and plants can not survive under these conditions of desert.However,in the desert ecosystem,as a small number of biological groups,microorganisms can survive tenaciously in the this ecosystem.In the desert ecosystem,as the dominant organism of the ecosystem,the microorganism is able to survive the high radiation,high oxidation and extreme drought in the desert,which shows that the desert microorganisms have formed the resistance mechanism in such an environment to protect its own survival and generation.Through the study on the mechanism of anti radiation and anti-oxidation of culturable microorganisms in desert,it can provide scientific basis and theoretical support for the resources of anti radiation and oxidative damage.In this paper,by culturable microorganisms in the Taklimakan Desert,the diversity of culturable microorganisms in the Taklimakan Desert was studied,and then by ways of UV-C andγray irradiation,the culturable bacteria in the Taklimakan Desert were screened for radiation resistance.The anti-oxidation research and metabonomics analysis of strain T1057 were figured out on the selected strong anti radiation strains and the resistence-radiation strains in Kumutag desert provided by the Key Laboratory of environmental microorganism resources and engineering of Gansu Province,Chinese Academy of Sciences.The results show that:1.A total of 2216 isolates were isolated from Taklimakan Desert,attributed to 4different phyla,respectively Actinobacteria,Firmicutes,Bacteroidetes and Proteobacteria,43 different genera and 352 different species.At the phylum level,Actinobacteria is the most dominant phylum from relative abundance among the culturable microorganisms in Taklimakan Desert.As well,Actinobacteria and Firmicutes are the dominant gates in CFU.2.In the Taklimakan desert from north to south,the distribution diversity of different species of microorganisms is different.At the gate level,TOC,TC,TN,WC,EC content and pH value were significantly correlated with the bacterial abundance of Actinobacteria,Firmicutes and Proteobacteria.Different environmental factors have significantly correlation effects on the distribution diversity and species diversity of bacteria in different phylum,and the ecological respond of this effect is different.3.The bacteria isolated from Taklimakan were irradiated by UV-C andγrays,and the bacteria with high radiation resistance were screened out.In addition to Bacteroides,there were 10 bacteria with stronger radiation-resistance than that of Deinococcus radiodurans,which were irradiated by HR(6000 J/m~2 UV-C).The results showed that:TKL1057(Kocuria indica),TKL798(Streptomyces griseoviridis),TKL860(Citricacus zhacaiensis),TKL1152(Planococcus citreus),TKL19(Gracilibacillus thalandensis),TKL588(Bacillus subtilis subsp.Inaquosorum),TKL606(Pseudomonas xanthomarina),TKL865(Paracoccus hibisci),TKL855(Pseudochrobactrum saccharolyticum)and TKL 35(Altererythrobacter soli),the radiation resistance of 10 strains was stronger than Deinococccus rRadiodurans.4.TKL1057(Kocuria indica)和TKL1152(Planococcus citreus),the ability of resistance-radiation of these two strains is stronger than Deinococccus Radiodurans,meanwhile,other strains’,except above two,survival rates tends to the value of 0.Thay means both TKL1057 and TKL1152 are the most resistant-radiation strains.5.Through antioxidant screening,we found that the antioxidant survival rate of different anti-radiation strains was TKL 1057>K81>TKL 1152>TKL 798>AQ(positive control)>k146>K202>TKL 865>k59>BL(negative control).At the same time,we also found that the concentration of resistant stress of different anti radiation strains was different.The antioxidant strains had good scavenging ability to DPPH,OH·and O~2-·radicals under oxidative stress,and the MDA content was also higher under oxidative stress.6.The metabolism of TKL1057(Kocuria indica)was determined after radiation and oxidation stress.It was found that after radiation and oxidation stress.The metabolites and metabolic pathways of TKL1057 have been done change significantly.After radiation,61 kinds of metabolites were expressed,40 kinds of metabolites were expressed after oxidation,40 kinds of metabolites expressed after radiation and oxidation were the same.At the same time,the analysis of foreign bodies in different treatments showed that the metabolism of cells after radiation was mainly based on the basic metabolism of sugar and secondary synthesis After oxidation treatment,organic acid is the main metabolite to synthesize secondary metabolites.更多还原