烟草叶片接种野火病菌后蛋白质表达差异

为了解烟草受野火病菌侵染后蛋白质表达变化情况,探讨烟草品种的抗病机制,本研究对烟草抗病品种‘K346’叶片进行人工接种,在接种后0～72h内分9次在接种点周围取无病原菌叶片组织提取叶片总蛋白,利用双向电泳技术分析接种处理叶片与未处理叶片的蛋白质表达差异,并选取6个减量表达蛋白点和4个增量表达蛋白点进行MALDI-TOF-MS质谱分析及数据库搜索鉴定,结果表明,6个减量表达蛋白包括参与植物呼吸代谢途径的ATP合成酶CF1α亚基、ATP合成酶CF1α链以及参与植物能量代谢循环途径的1,5-二磷酸核酮糖羧化酶/加氧酶相关蛋白;4个增量表达蛋白包括参与植物氨基酸代谢途径的谷氨酰胺合成酶、参与糖类和碳循环相关途径的高等植物光系统II氧复合物中Psbp A链蛋白、叶绿体景天庚酮糖-1,7-二磷酸酯酶和光捕获叶绿素a/b结合蛋白。经过蛋白质功能分析,推测烟草是通过加速植株生长、促进抗病相关糖和蛋白的表达、增强氨基酸代谢以及植株自身蛋白的修复等方式来提高抗病性。     

外源茉莉酸甲酯诱导的水稻叶片蛋白质差异表达分析

 以抗稻瘟病水稻近等基因系C101LAC及CO39为材料,应用差异蛋白组学技术对外源茉莉酸甲酯（MeJA）诱导8 h和12 h后的水稻叶片蛋白质差异表达变化进行了分析。采用PEG 4000预沉淀法提取水稻叶片总蛋白,经双向电泳、凝胶染色和图像分析,结果表明：与对照相比MeJA处理后的2个水稻品系中共有21个蛋白质表现为2倍以上的表达差异。其中,MeJA处理8 h后,CO39中的差异表达蛋白质点有5个,C101LAC中有8个,U5为2个品系中均新增的蛋白质点。MeJA处理12 h后,CO39中有6个差异表达蛋白质点,C101LAC中则有5个。经胶内酶解、MALDI TOF/TOF质谱分析及数据库检索,成功对21个差异表达蛋白质进行了鉴定,其中新增的蛋白质点U5被鉴定为内切β 1,3 1,4 葡聚糖酶。依据GO数据库和UniProtKB数据库提供的蛋白质注释信息,按照功能可将21个差异表达蛋白质分为8类,分别在植物抗性、氨基酸代谢、信号转导、光合作用、光呼吸、糖代谢、蛋白质合成与分解及细胞代谢等方面发挥作用。     

尖镰孢胁迫下黄瓜茎部蛋白质差异表达分析

为揭示尖镰孢胁迫下黄瓜茎部蛋白质差异,以黄瓜枯萎病高抗品系‘D0327’和感病品系‘649’为试验材料,分别接种2个毒力不同的尖镰孢C9和S1菌株后提取黄瓜茎部蛋白,运用双向电泳技术研究了尖镰孢侵染后茎部蛋白质组的变化。结果表明,2个尖镰孢菌胁迫下,抗感材料茎部蛋白表达发生了变化。茎部蛋白质双向电泳图谱与对照图谱进行比较分析,成功鉴定出8个差异蛋白点。强毒力菌株C9胁迫下,感病品系中鉴定出3个差异蛋白,分别是烯醇化酶及亚基、肌动蛋白和放氧增强蛋白,抗病品系中差异蛋白为烯醇化酶及亚基、磷酸丙糖异构酶和核苷酸二磷酸激酶。弱毒力菌株S1胁迫下,感病品系中差异蛋白为Csf-2蛋白,抗病品系中差异蛋白为放氧增强蛋白。这些蛋白分别参与能量代谢、光合作用和胁迫反应。     

不同供氮水平对芸豆碳氮代谢平衡的影响

采用盆栽土壤基施0、15、30 kg·hm-2三个氮素水平试验,研究了芸豆荚果形成后不同氮素水平下叶片和荚果的碳、氮代谢主要酶活性以及抗氧化酶系活性的变化。结果表明,芸豆荚果形成后,随着土壤供氮水平的增加,荚果的磷酸烯醇式丙酮酸羧化酶活性、多酚氧化酶活性、超氧化物歧化酶和过氧化氢酶活性逐渐增强;叶片的蔗糖合成酶活性、蔗糖磷酸合成酶活性、硝酸还原酶和磷酸烯醇式丙酮酸羧化酶活性大于同一供氮水平荚果的活性;且荚果的总可溶性糖和可溶性蛋白含量高于叶片;叶片的磷酸烯醇式丙酮酸羧化酶的活性与荚果的蔗糖磷酸合成酶和硝酸还原酶活性呈显著相关性。由此可以得出,源器官是芸豆植株的代谢活跃中心,而库器官成为植株的生长中心,且后者的碳、氮代谢过程主要受前者的调控。     

苹果酸脱氢酶与异柠檬酸脱氢酶在滞育七星瓢虫中的差异表达

为分析七星瓢虫滞育相关蛋白质的类别和功能,在滞育调控及滞育后生物学研究的基础上,应用双向电泳、质谱分析(ESI-QUAD-TOF)、生物信息学等蛋白质组学方法,对表达量存在2倍及以上差异且差异显著的蛋白点进行鉴定。应用Mascot软件进行数据库检索,根据数据的匹配程度鉴定蛋白质。所得蛋白质中,参与三羧酸循环的两种关键酶呈现差异性表达,其中苹果酸脱氢酶(gi|212508346)呈上调表达,异柠檬酸脱氢酶(gi|21392222)则呈下调表达。苹果酸脱氢酶的增加,推测与滞育状态下的生理需求相关:一方面与昆虫对NAD的合成和利用有关,另一方面或是七星瓢虫应对滞育环境条件的一种应激方式,与正常个体的代谢通路相比,滞育个体开启了另外的代谢通路以适应环境条件的改变。异柠檬酸脱氢酶作为三羧酸循环中起关键调节作用的限速酶,在滞育个体中下调表达,或反映了三羧酸循环整体速率的降低,表现在滞育七星瓢虫体内维持了低水平的能量代谢。本文的研究结果,为从蛋白质水平深入揭示七星瓢虫滞育调控及其机理提供一定的参考。     

抗感褐飞虱水稻遗传群体差异蛋白质组分析

为明确抗感褐飞虱Nilaparvata lugens水稻遗传群体在细胞水平的生理变化差异,通过蛋白质组学法对抗褐飞虱水稻遗传群体16W19-1-1和感稻飞虱水稻遗传群体16W45水稻叶片的差异蛋白质组进行研究,采用串联质谱标签标记和液相色谱-串联质谱联用仪进行差异蛋白质谱分析,利用Mascot search results软件进行搜库以确定差异表达蛋白,应用qPCR技术对差异蛋白关联基因在抗感褐飞虱遗传群体中的表达情况进行验证分析。结果表明,从抗感褐飞虱遗传群体中共鉴定出7 625个蛋白,其中差异蛋白229个,涉及上调蛋白156个,下调蛋白73个,主要集中在代谢蛋白、氧化还原蛋白和应激蛋白,主要参与了氰胺基酸代谢、牛磺酸代谢、聚糖降解、脂肪酸链伸长等通路。最终找到9个关键蛋白,关联6种酶,分别为γ-谷氨酰基转移酶、β-葡糖苷酶、β-N-乙酰-D-氨基葡萄糖酶、谷氨酸脱羧酶、甚长链烯酰-CoA还原酶和甚长链3-氧酰辅酶,对应8个基因。表明以上6种酶与植物抗虫性关系密切,可能在上述水稻品系抗褐飞虱中起着重要作用。     

灰葡萄孢蛋白激发子BcGs1诱导番茄抗病性及功能结构域鉴定

病原菌与植物互作过程中分泌的细胞壁降解酶具有致病和激发植物免疫的双重功能。BcGs1是从灰葡萄孢菌代谢物中分离的葡聚糖-1,4-α糖苷酶,能激发番茄和烟草的免疫防御反应,提高抗病性。BcGs1包含糖苷水解酶家族15(GH15)和糖结合模块家族20(CBM20)两个结构域,其激发植物防御反应的作用方式及功能结构域尚不清楚。本文测定了BcGs1不同诱导时间对提高番茄抗灰葡萄孢菌的效果、体外糖苷酶活性,并比较了BcGs1不同结构域截短蛋白与全长蛋白引起细胞坏死活性的差异。结果表明,BcGs1蛋白诱导番茄72 h可将灰葡萄孢菌引起的病斑面积减少23.25%;BcGs1能水解淀粉和海带多糖,但不能水解羟甲基纤维素和微晶纤维素;BcGs1全长、GH15和CBM20截短蛋白均能在烟草细胞中正常瞬时表达,但表达GH15的烟草叶片枯斑面积明显小于表达全长蛋白BcGs1的枯斑面积,表达CBM20截短蛋白的叶片没有产生枯斑反应;为了进一步明确截短蛋白GH15和全长蛋白BcGs1激发烟草细胞坏死活性的差异,用大肠杆菌表达并获得了纯化的重组蛋白GH15,叶片浸润法测定结果表明,截断蛋白GH15的细胞坏死活性仍然低于BcGs1全长蛋白的坏死活性,说明BcGs1诱导烟草细胞坏死活性需要GH15和CBM20结构域的共同参与。本文研究结果为进一步探讨BcGs1诱导植物抗病机制奠定了基础。     

水稻条斑病菌异柠檬酸脱氢酶在致病性中的功能分析

 异柠檬酸脱氢酶(isocitrate dehydrogenase,IcdH)催化异柠檬酸转化成α-酮戊二酸,参与碳代谢途径末端的三羧酸(tricarboxylic acid,TCA)循环。然而,编码异柠檬酸脱氢酶基因是否参与水稻条斑病菌(Xanthomonas oryzae pv. oryzicola,Xoc)的致病性,我们并不清楚。为了阐明IcdH的作用,通过同源重组技术获得了Xoc的icdH基因缺失突变体(RΔicdH),并对该突变体进行了相关功能研究。研究表明:该突变体不能利用苹果酸、丙酮酸和柠檬酸,在寄主水稻上的生长能力和致病力相对于野生型均显著降低,其游动性也显著减弱; 功能互补子恢复RΔicdH的上述表型至野生型水平; Real-time PCR结果显示,六碳单糖、蔗糖、苹果酸、丙酮酸与柠檬酸能显著诱导icdH基因的转录表达;与水稻细胞互作时icdH基因受诱导表达,并受HrpX和HrpG负调控。这些结果说明:icdH基因是Xoc获取碳源和在寄主水稻上具有致病性所需的。     

烯丙苯噻唑和氟唑环菌胺复配颗粒剂研究及其对水稻苗期稻瘟病和纹枯病的防效

水稻苗期病害的发生及防治备受关注。为明确诱抗剂烯丙苯噻唑与杀菌剂氟唑环菌胺复配颗粒剂对水稻幼苗的安全性和对苗期病害的防治效果,研制了16%烯丙苯噻唑·氟唑环菌胺复配颗粒剂配方并检测了理化性质,采用高效液相色谱法检测了有效成分含量及其缓释效果,并测定了温室条件下烯丙苯噻唑对水稻幼苗的安全性,以及颗粒剂对水稻稻瘟病和纹枯病的防治效果。结果表明,所制备颗粒剂的各项指标均符合国家相关标准要求。释放动态结果显示:杀菌剂氟唑环菌胺在V甲醇:V水=30:70的混合溶液中释放4 h后,累积释放率(质量分数)即达到峰值33%,且随后趋于稳定;而诱抗剂烯丙苯噻唑则在7 d后累积释放率方达到最高峰值;将两种有效成分复配可实现药剂对作物苗期病害的速效性与持效性协同作用。安全性试验显示,16%烯丙苯噻唑颗粒剂制剂用量不高于150 g/m2时对水稻幼苗安全。人工接菌条件下,试验剂量的烯丙苯噻唑可诱导水稻幼苗产生稳定的抗稻瘟病和纹枯病效果。16%烯丙苯噻唑·氟唑环菌胺颗粒剂对稻瘟病和纹枯病的防治效果在制剂用量150 g/m2剂量下最佳,分别为64.79%和68.32%,实现了1次用药同时防治两种病害的效果。研究结果可为诱抗剂与杀菌剂复配制剂的研发提供理论依据,同时可为缓释颗粒剂的田间科学施用提供参考。     

激发素与植物互作及抗病防卫信号传导

激发素是一类由疫霉属Phytophthora等真菌分泌的,可诱导茄科、十字花科等植物过敏性反应和系统获得抗病性的蛋白类激发子.激发素与植物的互作系统为研究植物抗病防卫信号传导途径提供了一个极好的模式.最近发现:激发素具有转移甾醇的活性,而形成甾醇和激发素复合物又是激发素与受体蛋白互作和激活抗病防卫信号传导途径的前提条件,也是对最近提出的病原激发子与受体互作"保卫假说"的支持.用125I标记法,找到了激发素的受体蛋白,由分子量约为160kD和50kD的2个亚基构成,被认为是Ca2 通道.在激发素激活的烟草抗病防卫信号传导途径中,蛋白质的磷酸化起着至关重要的作用,抑制蛋白激酶的活性,可阻止离子交换、活性氧进发、植保素合成等多种防卫反应;Ca2 参与诱导活性氧进发和植保素合成,活性氧进发与细胞死亡相关连,而植保素的合成与活性氧进发无关,过敏性反应对抗病防卫反应的建立不是必需的.激发素抗病基因工程在广谱抗病育种中具有广泛的应用前景.     

苹果枝条表皮应答轮纹病菌侵染的蛋白质组学分析

 蛋白质提取方法是双向电泳分析的关键。本研究中,我们通过优化提取条件,建立了适于苹果枝条表皮总蛋白提取的技术体系。在此基础上,开展了苹果枝条表皮应答轮纹病菌侵染的蛋白质组学研究,以期明确轮纹病菌胁迫下苹果枝条表皮细胞参与抗病反应的关键蛋白。具体方法是以未接菌及接菌后的苹果枝条表皮为材料,分别提取总蛋白,利用双向凝胶电泳技术结合质谱(MALDI-TOF-TOF/MS)检测分析差异表达蛋白。经质谱检测及数据库检索,共有22个蛋白点得到成功鉴定,按照功能划分为光合作用相关、糖类及能量代谢相关、防御反应相关、蛋白质合成相关及未知功能蛋白等5类。其中参与防御反应的病程相关蛋白(APX、 β-1,3-葡聚糖酶、Mal d1、PR-1)可能是苹果枝条表皮细胞应答轮纹病菌胁迫过程中参与抗病反应的关键蛋白。     

Comparative studies on carbofuran-induced changes in some cytoplasmic and mitochondrial enzymes and proteins of epigeic, anecic and endogeic earthworms

Exposure of epigeic (Perionyx sansibaricus), anecic (Lampito mauritii) and endogeic (Metaphire posthuma) earthworms to four different concentrations (10 ppm, 20 ppm, 40 ppm and 80 ppm) of carbofuran for 16 days induced specific activities of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH). P. sansibaricus showed 33-52% increase in specific activities of aerobic (cMDH, mMDH) and anaerobic (LDH) enzymes and proteins (cytoplasmic and mitochondrial). In L. mauritii, the enhancement in enzyme and protein level was 30-46%. Similarly, M. posthuma indicated 29-43% increase in profile of enzymes and proteins. The significant increase was observed on 2nd or 3rd day and it was maximum on 16th day of carbofuran exposure. Maximum effect of carbofuran was on epigeic earthworm. The sensitivity of mitochondrial dehydrogenase and protein of different earthworm species to carbofuran appears to be associated with oxygen availability in different strata of soil habitats. Inductions in enzyme specific activity and protein content were concentration and time-dependent. The effect was more pronounced on aerobic enzymes (cMDH, mMDH) as compared to anaerobic (LDH) one. The increases in mitochondrial enzyme (47%) and protein (43%) were more than the cytoplasmic enzymes (35%) and protein (31%), which suggest a greater effect of carbofuran on respiratory metabolism of earthworms. The carbofuran-dependent increase in cytoplasmic and mitochondrial enzymes and proteins might be due to increased synthesis of metabolic enzymes and stress proteins.     

Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin

Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.     

表达的hrmA蛋白质具有诱导水稻细胞产生防卫反应的活性

 将丁香假单胞菌hrmA基因构建到原核表达载体pET 29中,得到重组载体pET hrmA。将重组载体转化大肠杆菌BL21(DE3),经IPTG诱导,获得了以包含体形式存在的融合蛋白。复性的融合蛋白能诱导水稻悬浮细胞的活性氧迸发,处理20min后达到峰值。Northern杂交结果表明hrmA蛋白能显著地诱导PBZ1基因在水稻悬浮细胞中表达,在所观察的时间内PBZ1基因表达丰度随处理时间延长而增加,用hrmA处理水稻植株,诱导了PAL基因的表达。实验结果表明hrmA融合蛋白具有激活水稻细胞防卫反应的活性。     

Proteomics-based analysis reveals that Verticillium dahliae toxin induces cell death by modifying the synthesis of host proteins

Verticillium dahliae is one of the most destructive soil-borne fungal pathogens that cause vascular wilt diseases in a wide range of important crop plants, including cotton. However, the mechanisms used by this pathogen to infect cotton have not been fully elucidated. In the present study, we first investigated changes in protein abundance during the initial interaction between cotton roots and V. dahliae. Among the proteins that were upregulated upon infection, some were related to reactive oxygen species (ROS); among those downregulated upon infection were proteins involved in normal metabolism or cell structure. Further experiments confirmed that a sudden release of ROS and cell death accompany V. dahliae infection in the cotton vasculature. Further analysis indicated that a culture supernatant of V. dahliae induced lesion formation in tobacco leaves; de novo protein synthesis not active gene expression was required for this induction. Lesion formation was dependent on the age of leaves, but neither the known ROS burst nor the ubiquitin/26S proteasome system are prerequisites.     

茄青枯拉尔氏菌Rs-T02在3,4,5-三羟基苯甲酸甲酯作用下的差异蛋白分析

为探明3,4,5-三羟基苯甲酸甲酯(MG)抑制茄青枯拉尔氏菌的机制,利用二维电泳技术,分析了MG作用下茄青枯拉尔氏菌Rs-T02菌株的差异蛋白。结果发现在MG作用下的菌体中检测出29个差异蛋白点。采用质谱技术鉴定出22个差异蛋白,其中新增蛋白11种、缺失蛋白5种、表达上调蛋白1种和表达下降蛋白5种。这些蛋白参与能量代谢、信号转导、物质运输和DNA修复等途径。我们推测,与能量代谢相关的新增蛋白spot 9和缺失蛋白spot 65可能与MG抑制茄青枯拉尔氏菌的机制有关。     

Comparative proteomic analysis of cucumber roots infected by Fusarium oxysporum f. sp. cucumerium Owen

Cucumber Fusarium wilt (CFW), caused by the soil-borne fungus Fusarium oxysporum f. sp. cucumerium, is a serious disease in cucumber (Cucumis sativus) production worldwide. For the efficient control of the pathogenic fungi, a better understanding of its interaction and associated resistance mechanisms at the molecular level is required. Here, we report a comparative proteomics analysis of total root protein isolated from infected cucumber root of susceptible bulk (SB) and resistant bulk (RB) of cucumber generation F2. Two-dimensional gel electrophoresis (2-DE) coupled with MS/MS approaches identified 15 over-accumulated proteins from the RB plants. Identified proteins are mainly involved in defense and stress responses, oxidation reduction, metabolism and transport and other process. These proteins are likely to be a part of resistance-related protein network, playing different roles in cucumber disease resistance. Three vital clues regarding wilt resistance of C. sativus are gained from this study. First, jasmonic acid and redox signaling components were found in response to F. oxysporum infection in resistant plants. Second, the LRR family protein may play an important role in the defense reaction against CFW. Third, biotic and abiotic stress-related proteins were induced by the CFW fungus F. oxysporum, indicating the activation of common stress pathway.