番茄幼苗盐胁迫响应蛋白质组分析

采用营养液栽培,以盐敏感型番茄品种M82为试材,利用双向电泳(2-DE)研究盐胁迫处理下幼苗叶片蛋白质的表达谱,并采用基质辅助激光解析飞行时间串联质谱(MALDI-TOF/TOF-MS)技术进行差异蛋白质的分离及质谱鉴定。结果表明:(1)盐胁迫处理下,利用2-DE获得差异显著蛋白点20个,其中17个蛋白质点丰度上调表达,3个蛋白质点丰度下调表达。(2)通过质谱分析和蛋白质NCBInr数据库检索,共鉴定出19个差异蛋白,分别为果糖-二磷酸醛缩酶、S-腺苷甲硫氨酸合成酶、甘油醛-3-磷酸脱氢酶等及3个功能未知蛋白;这些鉴定出的差异蛋白质与能量代谢、光合作用、蛋白合成、氧化还原平衡等过程相关,暗示所分离鉴定的蛋白可能参与了番茄的盐胁迫响应,为进一步研究番茄抗逆机制奠定基础。     

蛋白质组学在研究植物响应逆境机理上的应用

逆境条件下植物可以通过改变其基因表达和相关代谢活动来适应,探讨植物基因和蛋白表达谱的变化就成为植物逆境响应机制研究中的重要内容,蛋白质表达谱反映了植物细胞和组织的实际状态,是植物基因表达和最终代谢的关键环节。随着蛋白质分离技术、质谱鉴定技术和植物生物信息学的迅速发展,蛋白质组学在植物响应逆境方面的研究中的应用已经比较成功,加深了人们对植物响应逆境机制的认识,并为人们提供了新的线索和思维。本文主要对蛋白质组学在植物响应非生物逆境(干旱、盐胁迫、低温胁迫、高温胁迫等)和生物逆境(病虫害)的机制研究的应用上进行了综述。     

蛋白质组学在植物逆境胁迫研究中的进展

蛋白质组学是后基因组时代研究的热点领域之一,自从蛋白质组这个概念被提出以来,其研究一直受到广泛关注,其研究技术也有了极大地进步。植物时刻都面临各种非生物胁迫,包括干旱、冷、盐、金属等,在长期进化过程中,植物形成独特的机制来响应逆境,然而目前对于植物如何适应逆境的分子机制尚未完全阐明。因此蛋白质组学作为一种强有力的研究技术手段,将为研究植物响应胁迫的分子机制提供理论支撑。介绍了蛋白质组学的产生背景、研究技术手段及植物在各种胁迫条件下的蛋白质组学研究、植物亚细胞器的蛋白质组学研究状况,同时对植物蛋白质组学的发展前景进行了展望。     

NaCl胁迫对小麦种子胚在萌发时期蛋白含量的影响

为比较不同浓度盐胁迫下小麦种子萌发前期胚蛋白表达情况,以"晋麦-47"为试验材料。选取经0、0.1和0.2mol/L处理过的种子,采用2-DE技术对3个浓度下胚蛋白含量变化进行研究。通过PDQuest图像分析软件分析盐胁迫下蛋白含量的变化,检测到正常的种子胚有121个点,0.1 mol/L NaCl胁迫下有32个蛋白点丰度下调,21个蛋白点丰度上调;在0.2 mol/LNaCl胁迫下,检测到46个蛋白质点丰度下调,16个蛋白点的丰度上调。研究结果表明,盐胁迫对小麦种子萌发时期胚中部分蛋白含量和表达产生不同程度的抑制与激活。     

NaCl胁迫对盐芥质膜和液泡膜ATPase活性的影响

以盐生植物盐芥和中生植物拟南芥幼苗为材料,研究了盐胁迫对它们叶片和根质膜、液泡膜H+-ATPase、Ca2+-ATPases和K+-ATPase活性以及H+-ATPase、Na+/H+ 逆向转运蛋白表达的影响.结果显示:在NaCl胁迫下,盐芥叶片和根质膜的H+-ATPase活性分别比对照显著升高41%～212%和35%～53%,液泡膜的H+-ATPase分别显著升高281%～373%和4%～38%,而拟南芥却比相应对照都显著降低;相同盐浓度胁迫下,盐芥叶片的H+-ATPase活性比根部高4～8倍,盐芥根也远高于拟南芥.在NaCl胁迫下,盐芥叶片和根的液泡膜H+-ATPase蛋白质β亚基含量变化与其酶活性变化趋势一致,质膜Na+/H+ 逆向转运蛋白的表达量与Na+含量变化趋势一致.盐胁迫下盐芥根中Ca2+-ATPases和K+-ATPase活性的增加与根中Ca2+和K+含量呈显著正相关.研究发现,在盐胁迫条件下,盐芥能有效增强H+-ATPase蛋白和Na+/H+逆向转运蛋白表达,显著提高其根系与叶片质膜和液泡膜的H+-ATPase、Ca2+-ATPase和K+-ATPase活性,维持细胞质中较高的Ca2+和K+水平,从而缓解盐胁迫的伤害,增强耐盐性.     

蛋白质组学研究揭示的甘蓝型油菜非生物胁迫应答机制

油菜(Brassica napus L.)是我国的主要油料作物之一,在生长发育过程中经常受到干旱、高温、高盐和营养缺乏等非生物胁迫。这些胁迫通常会阻碍油菜的生长发育,导致品质和产量下降。近年来,快速发展的高通量蛋白质组学技术为揭示油菜胁迫响应分子机制提供了新线索。本文综合分析了油菜不同组织/器官(如:叶片、根、下胚轴和种子)在响应盐、高温、干旱、草酸和缺素(磷、硫和硼)等逆境过程中675种蛋白质的丰度变化特征,揭示了其胁迫应答机制,主要包括:(1)通过G蛋白介导的信号通路感知与传递胁迫信号;(2)通过改变参与糖类与能量代谢相关酶的丰度调节代谢水平;(3)通过叶绿素合成的变化调节光合作用;(4)调节转录因子、蛋白质合成与命运相关蛋白质的丰度,从而在转录、翻译以及翻译后修饰等水平上应答逆境;(5)通过调节膜联蛋白、V型H+-ATP酶等质膜蛋白质,促进细胞内物质吸收与转运;(6)通过细胞骨架动态重塑保持正常细胞结构;(7)利用调节抗氧化酶系统清除活性氧,并通过合成多种防御物质减轻细胞受到的伤害。本综述为解析油菜逆境应答网络体系中的关键调控及代谢通路的变化提供了重要信息。     

植物水通道蛋白的干旱应答机制研究进展

干旱胁迫是严重影响全球作物生产的非生物胁迫之一,研究植物耐旱机制已成为一个重要领域。水通道蛋白是一类特异、高效转运水及其它小分子底物的膜通道蛋白,在植物中具有丰富的亚型,参与调节植物的水分吸收和运输。近10年来,水通道蛋白在植物不同生理过程中的作用,一直受到研究人员的关注,特别是在非生物胁迫方面,而研究表明水通道蛋白在干旱胁迫下对植物的耐旱性起着至关重要的作用,能维持细胞水分稳态和调控环境胁迫快速响应。水通道蛋白在植物耐旱过程中的调控机制及功能较复杂,而关于其应答机制和不同亚型功能性研究的报道甚少。该文综述了植物水通道蛋白的分类、结构、表达调控和活性调节,分别从植物水通道蛋白响应干旱表达调控机制、水通道蛋白基因表达的时空特异性、水通道蛋白基因的表达与蛋白丰度,水通道蛋白基因的耐旱转化四个方面阐明干旱胁迫下植物水通道蛋白的表达,重点阐述其参与植物干旱胁迫应答的作用机制,并提出水通道蛋白研究的主要方向。     

木薯叶片响应干旱胁迫的磷酸化蛋白质组差异分析

本文以抗旱性较强的‘华南8号’木薯(Manihot esculenta)为材料,分析正常供水、轻度干旱(干旱处理5 d)和重度干旱(干旱处理15 d)胁迫对木薯植株形态及叶片磷酸化蛋白质组的影响。结果表明,随着干旱胁迫程度的增加,木薯叶片从底部开始萎蔫、脱落,但顶端叶片优先保持正常生长。干旱胁迫处理后,共有28个磷酸化蛋白点在叶片中的表达丰度发生了显著变化。质谱(MS)鉴定显示,这些蛋白质主要参与光合作用、能量代谢、碳代谢、胁迫与防御、结合和转录翻译等代谢途径。其中,大部分参与光合作用的蛋白积累量在干旱胁迫后显著降低,而参与能量代谢、碳代谢、胁迫与防御、转录翻译等途径的大部分蛋白质积累量则明显升高。由此推测,木薯应答干旱胁迫可能是通过改变植株形态,抑制叶片中的光合作用相关蛋白,调控叶片碳分配过程,同时,通过有效清除活性氧,防御氧化胁迫损伤,防止蛋白变性和降解等方式。     

盐芥EsABCG25的克隆及其表达分析

旨在揭示盐芥(Eutrema salsugineum)ABCG25的结构及其对干旱、盐害以及温度等胁迫的响应。以山东生态型盐芥作为材料,通过电子克隆以及RT-PCR技术,获得一个腺苷三磷酸结合盒转运蛋白G(ABCG)家族成员基因的全长cDNA序列,命名为EsABCG25,GenBank登录号为KY111263。EsABCG25全长2 154 bp,含1 983 bp的开放阅读框,编码一个含有660氨基酸的蛋白分子。该基因定位于盐芥第5染色体,含有4个外显子和3个内含子。EsABCG25蛋白具有一个核苷酸结合结构域(NBD)和一个跨膜结构域(TMD),与来源于同科植物芜菁的ABCG25亲缘关系最近。对高通量测序数据分析表明该基因在盐芥的茎部表达最为丰富,在莲座叶中表达丰度最低,且在不同组织中均有可变剪接事件。Real-time PCR结果显示,该基因表达受多种逆境影响,尤其受到ABA的明显诱导。EsABCG25具有ABCG家族的典型序列特征;该基因在盐芥不同组织中的表达丰度存在差异,表达受到多种逆境的诱导,与盐芥的抗逆性关系密切。     

植物响应非生物胁迫的磷酸化修饰组学研究进展

植物具有固着生活的特点,高温、低温、干旱和盐等生境中常见的非生物胁迫会严重影响植物的生长发育。蛋白质磷酸化是植物应对非生物胁迫的重要机制,主要通过蛋白质的磷酸化和去磷酸化修饰来调控植物细胞对外界胁迫的应激反应,在植物细胞快速传递胁迫信号并激活对胁迫环境的形态、生理和分子水平适应机制的过程中起重要作用。该文主要介绍了植物磷酸化蛋白质的富集、检测和鉴定技术,并对近年来国内外有关植物响应高温、低温、干旱、淹水、盐、养分亏缺和元素毒害等非生物胁迫的磷酸化修饰蛋白组学研究进展进行综述。     

Proteomic analysis of cold stress-responsive proteins in <Emphasis Type="Italic">Thellungiella</Emphasis> rosette leaves

Low temperature is one of the most severe environmental factors that impair plant growth and agricultural production. To investigate how Thellungiella halophila, an Arabidopsis-like extremophile, adapts to cold stress, a comparative proteomic approach based on two-dimensional electrophoresis was adopted to identify proteins that changed in abundance in Thellungiella rosette leaves during short term (6 h, 2 and 5 days) and long term (24 days) exposure to cold stress. Sixty-six protein spots exhibited significant change at least at one time point and maximal cold stress induced-proteome change was found in long-term cold stress group while the minimal change was found in 6-h cold treatment group. Fifty protein spots were identified by mass spectrometry analysis. The identified proteins mainly participate in photosynthesis, RNA metabolism, defense response, energy pathway, protein synthesis, folding and degradation, cell wall and cytoskeleton and signal transduction. These proteins might work cooperatively to establish a new homeostasis under cold stress. Nearly half of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology suggesting that the cold stress tolerance of T. halophila is achieved, at least partly, by regulation of chloroplast function. All protein spots involved in RNA metabolism, defense response, protein synthesis, folding and degradation were found to be upregulated markedly by cold treatment, indicating enhanced RNA metabolism, defense and protein metabolism may play crucial roles in cold tolerance mechanism in T. halophila.     

Identification of changes in Triticum aestivum L. leaf proteome in response to drought stress by 2D-PAGE and MALDI-TOF/TOF mass spectrometry

Drought is an abiotic stress that strongly influences plant growth, development and productivity. To gain a better understanding of the drought-stress responses at physiological and molecular level in wheat plants (Triticum aestivum cv. KTC86211), we performed a comparative physiological and proteomics analysis. Eight-day-old wheat seedlings were treated with polyethylene glycol-simulated drought stress for 0, 24, 48 and 72 h. Drought treatment resulted in alterations of morphology, increased relative electrolyte leakage and reduced length and weight on leaf and root. Stress-induced proteome changes were analyzed by two-dimensional gel electrophoresis in conjunction with MALDI-TOF/TOF. Twenty-three spots differed significantly between control and treated plants following 48 h of drought stress, with 19 upregulated, and 4 downregulated, in leaf tissues. All of the differentially expressed protein spots were identified, revealing that the majority of proteins altered by drought treatment were involved in reactive oxygen species scavenging enzymes and photosynthesis. Other proteins identified were involved in protein metabolism, cytoskeleton structure, defense response, acid metabolism and signal transduction. All proteins might contribute cooperatively to reestablish cellular homeostasis under drought stress. The present study not only provides new insights into the mechanisms of acclimation and tolerance to drought stress in wheat plants, but also provides clues for improving wheat’s drought tolerance through breeding or genetic engineering.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated leaf disc transformation of <Emphasis Type="Italic">Thellungiella halophila</Emphasis>

Thellungiella halophila is a salt-tolerant close relative of Arabidopsis, which is adopted as a halophytic model for stress tolerance research. We established an Agrobacterium tumefaciens-mediated transformation procedure for T. halophila. Leaf explants of T. halophila were incubated with A. tumefaciens strain EHA105 containing a binary vector pCAMBIA1301 with the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, leaf explants were cultured on selective medium containing 10 mg l⁻¹ hygromycin and 500 mg l⁻¹ cefotaxime. Hygromycin-resistant calluses were induced from the leaf explants after 3 weeks. Shoot regeneration was achieved after transferring the calluses onto fresh medium of the same composition. Finally, the shoots were rooted on half strength MS basal medium supplemented with 10 mg l⁻¹ hygromycin. Incorporation and expression of the transgenes were confirmed by PCR, Southern blot analysis and GUS histochemical assay. Using this protocol, transgenic T. halophila plants can be obtained in approximately 2 months with a high transformation frequency of 26%.     

牵牛胁迫应答基因PnLOG2的克隆及功能验证

RING型E3泛素连接酶在植物应答非生物胁迫过程中发挥着重要功能。该研究从圆叶牵牛中克隆出RING型E3泛素连接酶基因PnLOG2,该基因序列号为XM_019321049.1。利用ORF Finder预测PnLOG2基因编码开放阅读框长度为912 bp (51~992 bp),编码313个氨基酸,蛋白分子质量34.38 kD,理论等电点为5.14。系统发育分析表明,PnLOG2基因与番茄亲缘关系最近。组织特异性分析表明,PnLOG2基因在牵牛不同组织均有表达,在老茎和新叶中表达量较高。qRT PCR分析结果表明,PnLOG2基因在圆叶牵牛根和叶中受干旱、盐碱胁迫诱导显著上调表达。通过异源表达PnLOG2基因于酵母细胞中,发现干旱、盐碱胁迫下PnLOG2基因提高了重组酵母的耐盐和耐旱能力,但降低了对碱的耐受性。该研究初步阐明了PnLOG2基因在干旱、盐碱胁迫下的功能,为进一步研究RING型E3泛素连接酶在非生物胁迫中的机理提供了理论依据。     

Proteomic Profiling and Protein-Protein Interaction Network Reveal the Molecular Mechanisms of Susceptibility to Drought Stress in Canola (<i>Brassica napus</i> L.)

Drought stress is one of the most important abiotic stresses that plants face frequently in nature. Under drought conditions, many morphological, physiological, and molecular aspects of plants are changed and as a result plants experience a remarkable reduction in growth, yield, and reproduction. To expand our understanding of the molecular basis of the plant response to drought stress, the proteomic profile and protein-protein network of canola (Brassica napus L.) were studied. The focus was to show molecular mechanisms related to canola susceptibility to drought stress. The experiment used a completely randomized design, implemented in a hydroponic system under greenhouse conditions. To impose drought stress, plants were exposed to Hoagland’s solution supplemented with polyethylene glycol (PEG) 6000 for 7 days. The drought stress resulted in 161reproducible protein spots in twodimensional electrophoresis of canola leaves. The t-student test showed 21 differentially abundant proteins (DAP), of which 2 and 19 were up and down accumulated, respectively. Two spots identified as 1-aminocyclopropane-1-carboxylate oxidase and D-2-hydroxyglutarate dehydrogenase showed an increased abundance of 2.11 and 1.77, respectively. The extended protein-protein interaction of differentially abundant proteins and KEGG analysis showed 47 pathways directly and indirectly associated with canola response to drought stress. DAPs with increased abundance were associated with amino acid and signaling processes, whereas DAPs with decreased abundance were mostly connected with pathways responsible for energy production. The results of the study will help to elucidate further the molecular events associated with the susceptibility to drought stress in canola.     

Comparative physiological,ultrastructural and proteomic analyses reveal sexual differences in the responses of Populus cathayana under drought stress

Drought is a major abiotic stress, limiting the survival and growth of young plants. However, little is known about sex‐dependent responses to drought at the proteome level. In this study, we carried out investigations on comparative proteomics, combined with physiological and organelle structure analyses, in males and females of Populus cathayana Rehd. Three‐month‐old poplar cuttings were treated at 30% of field capacity and at 100% field capacity as a control in a greenhouse for 40 days. Drought greatly inhibited plant growth, damaged the photosynthetic system and destructed the structures of chloroplasts, mitochondria and cellular membranes. However, males suffered less from the adverse effects of drought than did females. Using 2‐DE, 563 spots were detected, of which 64 spots displayed significant drought effect and 44 spots displayed a significant sex by drought interaction effect. The results suggest that the different responses to drought stress detected between the sexes have a close relationship to the changes in the expression of sex‐dependent proteins, including, e.g. photosynthesis‐related proteins, homeostasis‐related proteins and stress response proteins. These proteins could contribute to a physiological advantage under drought, giving potential clues for understanding sexual differences in the performance of plants in different environments.     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Functional screening for salinity tolerant genes from <Emphasis Type="Italic">Acanthus ebracteatus</Emphasis> Vahl using <Emphasis Type="Italic">Escherichia coli</Emphasis> as a host

Salinity reduces plant growth and crop production globally. The discovery of genes in salinity tolerant plants will provide the basis for effective genetic engineering strategies, leading to greater stress tolerance in economically important crops. In this study, we have identified and isolated 107 salinity tolerant candidate genes from a mangrove plant, Acanthus ebracteatus Vahl by using bacterial functional assay. Sequence analysis of these putative salinity tolerant cDNA candidates revealed that 65% of them have not been reported to be stress related and may have great potential for the elucidation of unique salinity tolerant mechanisms in mangrove. Among the genes identified were also genes that had previously been linked to stress response including salinity tolerance, verifying the reliability of this method in isolating salinity tolerant genes by using E. coli as a host.