活性氧在酿酒酵母乙醇胁迫中的作用

I摘要酿酒酵母是一种传统的乙醇生产微生物，通常用于乙醇燃料生产和酿造业，但过量的乙醇会抑制细胞的生长和活性，对酵母细胞产生毒害作用。ROS（ReactiveOxygenSpecies）是一类性质活泼的小分子化合物，在多种逆境胁迫中期起重要作用，但是否在乙醇胁迫中起重要作用尚无定论。为了解ROS在乙醇胁迫中的作用及机制，本文以酿酒酵母为材料，检测了乙醇胁迫对酵母细胞超氧阴离子（O2·-）含量、过氧化氢（H2O2）含量、酵母细胞生长状况、膜完整性、线粒体膜电位以及脂质和蛋白质损伤的影响。实验结果表明，乙醇可以抑制对数生长期酵母的生长，导致膜完整性受损、线粒体膜电位降低、O2·-和H2O2水平增加、脂质过氧化物MDA含量增加。加入外源还原型谷胱甘肽（GSH），蛋氨酸（Met），N-acetylcysteine（N-乙酰半胱氨酸，NAC）后，可以提高酿酒酵母细胞存活率及膜完整性，线粒体膜电位、降低O2·-和H2O2水平。添加GSH，Met，NAC到发酵培养基中，检测发酵后乙醇含量发现，正常组为7.3%，Met，NAC可以将乙醇产率分别提高到8.62%和8.56%，而GSH只有6.35%。以上结果表明乙醇能够诱导O2·-和H2O2等ROS的产生，导致氧化胁迫，从而造成酵母细胞发生膜质过氧化，生长受到抑制。而GSH，Met，NAC可以清除过量的ROS，降低乙醇胁迫引起的酵母损伤，其中Met和NAC虽然可以提高乙醇产率，但是只有NAC处理在统计学上具有差异性。为进一步研究ROS变化的机制，采用自噬抑制剂3-MA和自噬诱导剂雷帕霉素(rapamycin)处理乙醇胁迫条件下的酵母细胞，检测对O2·-和H2O2水平，菌落形态以及膜完整性的影响。结果表明，3MA可以有效降低O2·-和H2O2水平，降低乙醇对酵母菌落的抑制，提高膜完整性，而雷帕霉素进一步加剧对菌落的抑制，降低膜完整性，提高O2·-和H2O2水平。表明自噬是影响ROS水平的机制之一，它通过调节ROS参与酿酒酵母乙醇胁迫损伤。关键词：酿酒酵母；乙醇胁迫；自噬；ROSIAbstractSaccharomycescerevisiaeisastrainusedtoproduceethanolandisappliedintheproductionofethanolfuelandbrewingindustry.Saccharomycescerevisiaeisatranditionalyieldethanolstrain,butissensitivetothehighconcentofethanol.Excessiveaccumulationofenthanolcouldinhibitthegrowthandactivityofcells,inducereactiveoxygenspecies，therebyleadtoadamagetoyeastcells.Autophagyisanimportantdegradationpathwayformaintainingthebalanceandmattercycleineukaryoticcells.Althoughwehaveacertainamiyntresearchonethanolstress,themechanismofethanolstressinSaccharomycescerevisiae,whichROSplaysaleadingroleunderethanolstress,whetherautophagyisinvolvedinethanolstreesandwhatroleautophagyhasinethanol.Theaboveproblemsarenotyetclear.TounderstandwhichROShasanimportantroleinethanolstress,SaccharomycescerevisiaefromAngelYeastwasastheexperimentalmaterialinthiswork,westudiedtheeffectofreactiveoxygenspecies(ROS)onSaccharomycescerevisiaegrowthandfermentationunderethanolstress,throughdetectingphysiologicalindicatiorsincludingcellgrowth,themembraneintegrity,superoxideanion(O2·-)conctent,hydrogenperoxide(H2O2)content,malondialdehyde(MDA),carbonylcontentandsoon.Theresultshowedthatethanolcouldinhibitthegrowthofyeastcellsinthelogarithmicphase,damagethemembranceintegrity,risethelevelsofO2·-andH2O2,increasethecontentofMDA(theproductofLipidperoxide).WeusedexogenousGlutathione(GSH),Methionine(Met)andN-acetylcysteine(NAC)treatedethanolstressedSacchayomycescerevisiaerespectively,andfoundthattheycouldreducedthelevelO2·-andH2O2,increasedcellsactivityanddecreasedtheconcentofMDA.ThisindicatesthaethanolcouldinducethegenerationofO2·-andH2O2,ledingtotheoxidativestress,resultinginthedamagetoyeastcells.Whenthegrowthinhibitedserious,ethanolevencouldleadtothedeath.GSHandMet,NACcouldcleartheexcessofROS,reducetheyeastdamagecausedbyethanolstressandimprovetheethanolyield.InordertounderstandwhetherautophagywasinvolvedintheethanolstressintheS.cerevisiaeaswellasitwasrelatedwithROS,weused3-MA(autophagyinhibitor)andrapamycin(autophagyinducer)totreatethanol-stressedSaccharomycescerevisiae,IIthemembraneintegritywasbyPIstaining,thelevelofO2·-andH2O2wasdetectedbyROSfluorescentprobeDHEandDCFH-DAstaining.Theresultsshowedthat3-MAcouldreducetheinhibitionofethanolonyeastcells,improvethemembaneintegrity,reducethelevelsofO2·-andH2O2.Butrapamycincouldfurtherdeepenthecolonyinhibition,reducethemembraneintegrity,improvethelevelsofO2·-andH2O2.ThisindicatesthatautophagyinvolvesinSaccharomycescerevisiaeethanolstressbyregulatingthelevelofROS,andplaysaroleofmaintainingcellsurvivalinethanol-stressedSaccharomycescerevisiae.Keywords:Saccharomycescerevisiae；ethanolstress；autophagy；ROSIII缩略词英文缩写英文全称中文名称BSABovineserumalbumin牛血清白蛋白CvtCytoplasmtovacuoletargeting细胞质到液泡途径DAB3,3´-Diaminobenzidine,tetrahydrochloride3，3’-二氨基联苯胺四盐酸盐DCFH-DA2′,7′-Dichlorodihydrofluoresceindiacetate2’，7’-二氯荧光素二脂DHEDihydroethidium二氢溴化非啶DMSODimethylsulfoxide二甲基亚砜DNPH2,4-Dinitrophenylhydrazine2,4-二硝基苯肼EDTAEthylenediaminetetraceticacid乙二胺四乙酸GSHGlutathione谷胱甘肽MetMethionine蛋氨酸3-MA3-Methyladenine3-甲基腺嘌呤NACN-acetylcysteineN-乙酰半胱氨酸NBTNitrobluetetrazolium氯化硝基四氮唑蓝O2·-Superoxideanion超氧阴离子PASThephagophoreassemblysite自噬体组装位点PBSPhosphatebuffersolution磷酸缓冲液PMSFPhenmethylsulfonylchlorine苯甲基磺酰氯ROSReactiveoxygenspecies活性氧rpmRoundsperminute每分钟转速SAMS-Adenosylmethionine,腺苷蛋氨酸SDSSodiumdodecylsulfate十二烷基硫酸钠TBA2-Thiobarbituricacid硫代巴比妥酸TCATrichloroaceticacid三氯乙酸Tris2-AminoAmino-2-(hydroxymethyl)-1,3-propanediol三羟甲基氨基甲烷Triton-X-100Triton-X-100曲拉通100IVV目录第一章前言...................................................................................................................11.1乙醇对酵母的影响....................................................................................................11.1.1乙醇对酵母的毒害作用..........................................................................................11.2活性氧........................................................................................................................21.2.1活性氧的危害.........................................................................................................31.2.2常见抗氧化剂ROS防御系统...........................................................................