【作者】 刘向勇；

【导师】 鲍晓明；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 在自然条件下,生物的生存依赖于对外界环境改变的及时识别以及对新环境所作出的相应反应。生物对不良环境刺激因子的应答反应,并最终形成对胁迫环境的抵抗力就是环境胁迫反应(environmental stress response,ESR)。生物胁迫反应是研究基因功能和了解生命活动本质的便利模型。同时,对生物胁迫反应的研究在培育抗逆植物、改造工业微生物的生产形状以及对细胞癌变的机理与治疗等方面的研究具有重要作用。酿酒酵母(Saccharomyces cerevisiae)是最简单和最早完成全基因组测序的真核生物(http://genome-www.stanford.edu/Saccharomyces)。酿酒酵母易培养、生活周期短,遗传背景清晰,同时以酿酒酵母为基础的分子生物学研究技术平台日趋完善,很容易制备各种突变体等遗传学研究材料,使研究方便可行,是理想的真核生物模式种。高盐环境一直是人们关注的重要的胁迫因子之一,以酿酒酵母为模型开展盐胁迫机理研究,在国际上一直是生物学研究的热点之一。目前,对酿酒酵母胁迫反应机制已经有了较深入的了解,高盐胁迫对酵母细胞的毒害作用包括两个方面,一是Na~+离子毒害;二是产生渗透压胁迫,使质膜的跨膜渗透压降低而导致细胞膨胀压的丧失。同时,一些重要的盐胁迫信号传导途径和离子通道蛋白相继被发现。例如:在高浓度NaCl条件下,高渗透性甘油促分裂原激酶信号转导途径(high osmolarity glycerol mitogen activated protein kinase signaling transductionpathway,HOG-MAPK)通过促进甘油积累抵抗渗透胁迫刺激。钙调磷酸酶(Calcineurin,CaN)信号途径通过转录因子Crzlp/Tcnlp激活酵母细胞ENA1(编码重要P-型ATPase离子外排泵)的表达,促进Na~+外排。同时TRK1/2p负责的K~+/Na~+离子吸收系统,通过提高K~+离子亲和性,优先积累K~+限制Na~+的吸收,减少对细胞的毒害作用Na~+。本课题组前期利用转座标签随机插入突变技术,首次在酿酒酵母中发现Bdf1p(Bromodomain Transcription Factor 1)转录因子与高盐胁迫有关。BDF1基因的缺失并不会引起致死效应,属于非基础性的基因转录调控因子。Bdf1p转录因子在蛋白质结构上包含:2个“溴”结构域(Bromodomain)和一个位于C末端的ET(Extra-terminal)结构域,属于BET转录因子家族。Bromodomain结构域是近10年来发现的广泛存在于多种真核生物中的组蛋白赖氨酸乙酰化位点结合结构域,由60～110个高度保守的氨基酸残基组成,目前对于含Bromodomain结构域蛋白功能的研究还处于起步阶段。本研究利用基因敲除(gene knockout)和化学药物阻断的方法,对盐胁迫条件下HOG-MAPK途径、Calcineurin途径、ENA1基因、以及TRK1基因与BDF1基因之间的遗传学关系进行了分析。实验结果表明,当BDF1基因分别与HOG1、CMP1CMP2、ENA1和TRK1基因同时缺失或阻断时,酵母细胞的盐耐受性均进一步减弱,该结果提示BDF1基因在酵母盐胁迫应答反应中与上述途径/基因可能分别涉及不同的酵母盐胁迫途径。基因表达谱分析技术为酵母盐胁迫反应分子机制及其相关基因功能的研究提供了全新和有效的研究手段。利用酵母全基因组芯片分别绘制了盐胁迫条件下野生型菌株和bdf1△菌株的基因表达谱,分析比较相互间的不同,在全基因范围内寻找受Bdf1p影响的盐胁迫反应相关基因。野生型菌株和bdf1△菌株细胞经0.6 M NaCl处理45 min后,提取酵母总RNA,合成cDNA并分别用Cy5-dCTP和Cy3-dCTP标记,与含有5935个开放阅读框(open reading frame,ORF)的酵母全基因组芯片进行杂交并扫描。采用GenePix Pro 4.0图像分析软件(AxonInstruments公司)对芯片图像进行分析,计算ratio值(两种荧光强度的比值cy5/cy3);然后对芯片上的数据用Lowess方法进行归一化;最后用T-test和以两倍差异(即ratio值大于等于2.0,小于等于0.5)的标准来确定差异表达基因。为了消除荧光偏向性带来的假阳性,盐处理和对照的酵母细胞RNA样品在反转录过程中进行荧光交换实验,同时对于每个菌株盐处理(W303盐处理/W303和bdf1△盐处理/bdf1△),都作两次独立的生物学重复实验,分析两次独立实验中表达变化趋势一样的基因,基因变化的数值取两组数据的平均值。在此基础上,随机挑选差异表达的基因,利用实时荧光定量RT-PCR(real-time quantitativeRT-PCR,qPCR)进行验证,实验结果显示对所测定的基因与基因芯片技术检测结果一致,不仅变化方向一致,而且表达值非常接近,说明芯片结果具有良好的准确性。本实验共筛选出差异表达基因217个,有144个差异表达基因分别在野生型菌株与bdf1△菌株呈现相同的变化趋势(上调基因119个,下调基因25个)。采用MIPS数据库的在线软件Functional Catalogue Database(http://mips.gif.de/proj/funcatDB/search_main_fra me.html)进行生物学功能分类对上述144个差异表达基因进行功能分类发现,以上差异表达基因主要集中在甘油代谢、海藻糖代谢、离子通道蛋白、氧化胁迫反应、热休克蛋白和功能未知蛋白等方面。其中ENA1基因在野生型菌株和bdf1△菌株中的盐胁迫诱导表达倍数相似(5.25和6.32),说明BDF1基因缺失并未对盐胁迫条件下ENA1基因的正常表达产生影响,与前面遗传学分析结果一致,即BDF1和ENA1基因分别涉及不同酵母盐胁迫途径。同时,基因芯片数据显示甘油合成关键酶基因GPD1(编码3-磷酸甘油脱氢酶),经盐胁迫处理后,在野生型菌株和bdf1△菌株的基因表达水平均显著上升,其诱导表达倍数分别高达35.79和35.52倍。酵母胞内甘油含量测定结果表明,经0.6 M NaCl处理45 min后,野生型菌株和bdf1△菌株中甘油积累均有大幅提高且含量相近,此结果与上述通过基因芯片在转录水平上测定结果一致。BDF2是酿酒酵母BDF1基因的同源基因,采用长臂同源多聚酶链式反应(LFH-PCR)方法制备含有遗传霉素耐药基因(kanMX4)的BDF2基因敲除片段并转化酵母,构建了bdf2△菌株。梯度生长实验表明,与野生型菌株相比,BDF2基因缺失并不造成盐敏感表型,说明BDF2基因是酵母盐抗性的非必须基因。基因芯片结果显示,BDF1基因缺失导致盐胁迫条件下BDF2基因表达下调,提示两基因间的表达可能存在相互影响,盐胁迫条件下两基因间的遗传学关系有待进一步研究。线粒体是真核生物能量代谢的中心,在物质代谢、细胞凋亡过.程中发挥了重要的作用。基因芯片结果显示,经0.6 M NaCl处理45 min后,与野生型菌株相比,BDF1基因缺失造成盐胁迫条件下大量线粒体功能相关基因表达下降。其中包括:线粒体核糖体基因(MRPL3、MRP4、MRP7和MRP11),细胞色素C基因CYC1,线粒体延伸因子基因MEF1和线粒体缬氨酰-tRNA合成酶基因VAS1,该结果提示,Bdf1p可能在维持盐胁迫条件下线粒体的正常功能方面发挥重要作用。线粒体膜电位(Mitochondrial membrane potential,△Ψm)和胞内活性氧(reactive oxygen species,ROS)含量是评价线粒体功能的的重要指标。利用还原型线粒体特异性荧光探针Mito Tracker Red CMXRos对盐胁迫处理后野生型菌株和bdf1△菌株中线粒体膜电位的变化进行了检测。实验结果表明,经0.6 M NaCl处理45 min后,Mito Tracker Red CMXRos探针在野生型菌株细胞中仍处于聚集状态,呈现网状分布的荧光线条,而大约20%左右的bdf1△菌株细胞呈现弥散状分布的荧光,说明这些细胞的线粒体跨膜电位下降或消失。利用DHR荧光探针(dihydrorhodamine 123),对经0.6 M NaCl盐胁迫处理45 min的野生型菌株和bdf1△菌株的胞内ROS含量进行了测定。实验结果表明,经盐胁迫处理后的野生型酵母细胞在荧光显微镜下呈现微弱红色荧光,而BDF1基因缺失菌株显现明亮的红色荧光,荧光强度相对定量分析表明,bdf1△菌株细胞的相对荧光强度比野生型菌株细胞高出1.5倍以上,说明BDF1基因缺失可导致盐胁迫条件下胞内活性氧ROS的积累。超氧化物歧化酶(superoxide dismutase,SOD)是酵母体内活性氧清除体系中最重要的抗氧化酶,酶活测定结果表明,0.6 M NaCl处理45min的盐胁迫条件下野生型菌株和bdf1△菌株中SOD酶活性均呈上升趋势,且bdf1△菌株的SOD酶活性比野生型菌株高出38个活力单位以上。该结果表明,盐胁迫条件下bdf1△菌株细胞中ROS的大量积累并不是由于超氧化物歧化酶的活性受到抑制引起的。细胞凋亡(apoptosis)是指细胞在一定的外界刺激或病理条件下,受基因控制的一种细胞自主的有序的死亡。线粒体功能紊乱与酵母细胞凋亡的发生密切相关。利用DAPI染色和透射电子显微镜(TEM)对盐胁迫处理后,野生型菌株和bdf1△菌株细胞的细胞核和染色质形态进行观察。结果显示,与野生型菌株相比,bdf1△菌株细胞的细胞核裂解,染色质发生凝集,核膜皱缩,呈现明显的细胞凋亡特征,说明盐胁迫条件下BDF1基因缺失可引发线粒体依赖的酵母细胞凋亡。更多还原

【Abstract】 Cellular organisms have developed several autonomous mechanisms to adjust themselves to new conditions,because they are constantly challenged by changing environmental conditions,.Under stress conditions,the cells undergoes a series of changes to protect the external system from the detrimental effects of stress,which were referred to as the environmental stress responses(ESR).The studies of yeast stress responses have provided a powerful tool for investigation of the genes function and elucidation of the nature of important life activities.In addition,research on stress response has important role for the improvement of the stress resistance of plant corps and industrial microbial,and has a large impact on medical issues.Saccharomyces cerevisiae was the first eukaryotic organism whose whole genome was completely sequenced(http://genome-www.stanford.edu/Saccharomyces).The rapid growth,ease of genetic manipulation,and a well-defined genetic system of the yeast Saccharomyces cerevisiae make it ideal for fundamental and applied studies in eukaryotic species.Salt-stress adaptation mechanisms are being intensively studied in model organisms Saccharomyces cerevisiae.Under high salt conditions,the yeast cells are mainly challenged by ion and hyperosmotic stress.Several mechanisms involved in yeast salt stress response have been identified.For instance,under NaCl stress,the high osmolarity glycerol mitogen activated protein kinase signaling transduction pathway (HOG-MAPK)is activated,which mediates the production and accumulation of the osmolyte glycerol.Calcineurin functions through the Crz 1 p/Ten 1 p transcription factor to induce several salt stress-responsive genes expression,such as the ENA1 gene which encodes a plasma membrane P-type Na~+-ATPase ion extrusion pump. Additionally,the TRK1/2p transport system increased affinity for K~+ and prevent entry of excess Na~+,to increase NaCl tolerance.Yeast mutants with impaired K~+ transport system showed increased sensitivity to salt stress due to accumulate of more Na~+ than wild type strain.The S.cerevisiae BDF1 gene,which encodes a bromodomain-containing transcription factor,was isolated in a large-scale screen for salt-sensitivity mutants following transposon mutugenesis.Bromodomain transcription factor 1 protein(Bdf1p)contains two bromodomains and an ET(extra-terminal)domain.It is a member of the BET protein family.In yeast, Bdf1p was not essential for cell viability.Bromodomains are evolutionarily conserved and act as acetryl-lysine binding domains that process the molecular information conveyed by lysine acetylation modification.To understanding the biological functions ofbromdomain-containing proteins in cellular processes were currently at a fledgling stage.By using gene knockout and drugs block techniques,the relationship between BDF1 and several signaling transduction pathways/genes have been known in salt tolerance,was investigated.These included HOG-MAPK pathway,Calcineurin pathway,ENA1 and TRK1 genes.Genetic analysis indicated that the function of Bdf1p in salt tolerance is independent of the HOG-MAPK pathway or the calcineurin pathway,K~+ transportation system and is not meditated by Enalp,the major determinant of Na~+ extrusion system.The yeast genome-scale cDNA microarray platform provides an effective research tool to investigated the global changes in gene expression under a given salt stress condition.To gain further insight into the role of Bdf1p in salt tolerance on a global scale,DNA microarray analysis was conducted.The global gene expression changes in the wild-type and a bdf1△mutant responding to a salt stress treatment(0.6 mol/L NaCl,45 min)were measured,respectively.Total yeast RNA was isolated after treated by0.6 mol/L NaCl for45 min,and the cDNA were labeled with Cy5-dCTP and Cy3-dCTP,respectively.Then the Cy5/cy3-1abeled cDNA were hybridized on the yeast genome-wide DNA microarrays containing 5935 ORFs.Obtained images were analyzed with a GenePix Pro 4.0.A space and intensity-dependent normalization based on the LOWESS program was employed.We determined the levels of gene expression based on the statistical method student’s t-test in combination with a 2-fold cutoff Genes whose ratio changed＞2-fold with p＜0.05 were determined to be have significantly different gene expression.To eliminate the dye-bias artifact,the dye-swap experiment was performed for two different RNA populations.And two biologically independent experiments of each of the samples(wild-type,wild-type salt treated,bdf1△,bdf1△salt treated)were analyzed.Only genes having consistently altered expression in two independent experiments were selected for further analysis,and the final fold changes were derived from the average of two independent experiments.Based on this experiment design,totally 217 differentially expressed genes were identified in salt-treated yeast cells(wild type and bdf1△mutant),in which 144 total genes(119 up-regulated genes and down-regulated 25 genes)displayed the same altered tendency in the wild type and bdf1△mutant after the salt treatment.Functional classification of the 144 differential expressed genes were base on the MIPS Functional Catalogue Database(http://mips.gif.de/proj/funcatDB/search_main_fra me.html).The above genes are associated with functions,such as glycerol metabolism,trehalose metabilism,ion transport,heat shock protein,unclassified protein,and so on.Interestingly,the expression level of ENA1 had a similar increase in the wild type and bdf1△mutant(5.25- and 6.32- fold,respectively),suggesting that the function of ENA1 in Na~+ tolerance is independent of BDF1.Additionally,The glycerol-3-phosphate dehydrogenase gene(GDP1),which is a key enzyme of glycerol metabolism,was highly induced in the wild type and bdf1△mutant(35.79- and 35.52- fold, respectively)after the salt treatment.The determination of intracellular content of the yeast cells showed that there was a similar level of glycerol in wild type and bdf1△mutant after the salt treatment,which is consistent with the data from the microarray analysis.Change in expressions of two randomly selected genes(MEF1 and YNL208w)were further confirmed by real-time quantitative RT-PCR.And the results suggested good agreement between the microarray and real-time quantitative PCR analysis. In yeast,BDF2 gene is a homolog of the BDF1 gene.Construction of TRK1 deletion mutants was performed by long flanking homology(LFH)-PCR and the kanMX4 deletion marker was used.In spot dilution growth assays,we have not observed a salt sensitive phenotype of the bdf2△mutant compared to the wild-type at various NaCl concentrations,which suggests that the BDF2 gene is not essential for salt resistance. In addition,the microarray results showed that,in response to the salt stress,the expression of BDF2 gene was down regulated in bdf1△mutant but not in the wild type strain.This result indicated that there is some genetic interaction between BDF1 and BDF2 gene,and the roles of Bdf1p and Bdf2p in salt tolerance remains to be determined.It has been known that the mitochondrial functions are important for energy metabolism and have an intimate relation with cell death.The microarray results showed that 7 genes associated with mitochondrial function were downregulated in the bdf1△strain but not in the wild type strain after the salt treatment(0.6M NaCl for 45min).These included four mitochondrial ribosomal genes(MRPL3,MRP4,MRPL7, MRPL11),Cytochrome c Oxidase encoding gene CYC1,a gene encoding a mitochondrial elongation factor MEF1and a gene encoding a mitochondrial valyl-tRNA synthetase VAS1.This result suggested a possible role for Bdf1p in the control of mitochondrial functions under salt stress conditions.Mitochondrial membrane potential(△Ψm)and reactive oxygen species(ROS) production are useful indicators of mitochondrial function.To measure△Ψ, Mitotracker red CMRos was used,which CMRos(Molecular Probes),which stains mitochondria in a△Ψm-dependent fashion.The MitoTracker specifically stains mitochondria when the△Ψm is high,but stains in a diffused pattern when△ΨM is lost.After treated with 0.6 M NaCl for 45 min,the wild type cells displayed a very nice,linear MitoTracker staining,but about 20%of bdf1△cells showed a diffused staining,indicating that absence of Bdf1p caused△Ψloss under salt stress conditions. To monitor the ROS production of yeast cells,we used dihydrorhodamine 123(DHR), which can be oxidized by the intracellular ROS to become fluorescent chromophore rhodamine.After treated with 0.6 M NaCl for 45 min,wild-type cells showed a very dim,red fluorescence after incubation with DHR,whereas bdf1△cells had intense red fluorescence staining with DHR after treatment with NaCl.Relative fluorescence intensities were further quantified on a fluorescent microplate reader.About 1.5-fold increase in fluorescence intensities was observed in the bdf1△mutant compare with the wild-type strain.Superoxide dismutase(SOD)is the key enzyme providing cells protection from the ROS toxicity.After the salt treatment,the SOD activity increased significantly in bdf1△,and was 38 unit higher than in wild type cells.This result indicated that the SOD activity responded normally in bdf1△mutant and the accumulation of ROS in bdf1△cells was not due to the inhibition of Superoxide dismutase activity in the mutant.Apoptosis is an intrinsic cell death process.Mitochondrial dysfunction has been described as a key event in triggering yeast apoptosis.DAPI staining and examination with transmission electron microscopy revealed that the salt-stress treated bdf1△cells displayed randomly distributed nuclear fragments,extensive chromatin condensation and margination along the nuclear envelope,which is a typical marker of apoptosis. This result indicated that absence of Bdf1p caused mitochondria-dependent yeast apoptosis under salt stress conditions.更多还原