基于iTRAQ定量蛋白质组技术筛选‘华月’苹果斑点落叶病抗性相关蛋白

 为了探讨苹果叶片抗病相关蛋白的表达特性,进一步解析苹果叶片自身防御反应分子机制,本文以抗病苹果品种‘华月’为试材,采用iTRAQ定量蛋白质组技术分析苹果链格孢菌处理前后,苹果叶片总蛋白差异表达情况。结果表明,与无菌水处理的叶片相比,病原菌接种后的叶片共筛选到433个差异表达蛋白,聚类分析结果较好,其中257个蛋白显著上调,176个蛋白显著下调。进一步开展差异蛋白筛选及功能分析,筛选后的53个蛋白依据功能可划分为7类,分别为代谢过程相关,防御或逆境应答反应相关,蛋白质代谢过程相关,细胞分裂相关,细胞壁修饰相关,细胞过程相关及其他功能未知蛋白。代谢通路分析表明,KEGG共富集到7个结果,64个蛋白在7条通路中得到注释,且这些蛋白对这7条信号通路均具有显著影响(P<0.05)。亚细胞定位分析结果表明,共49个蛋白得到成功定位,其中35个蛋白定位于叶绿体,表明叶片细胞中大量的叶绿体蛋白参与了病原菌胁迫应答反应。其他蛋白中,7个定位于细胞质,3个定位于细胞质膜,其余4个分别定位于细胞壁、胞外、过氧化物酶体及内质网。除了防御或逆境反应相关蛋白,光合作用及其他代谢相关蛋白也参与了苹果叶片细胞的防御反应。病程相关的PR类蛋白中包括3个过氧化物酶类蛋白(PR-9)、1个类甜蛋白(PR-5)及1个MLP样蛋白(PR-10),过敏反应相关的MLP样蛋白在果树应答逆境胁迫的研究中的报道较少,MLP蛋白的差异表达可能也与苹果叶片的防御反应相关。本研究进一步证实了苹果叶片应答链格孢菌胁迫的抗性相关蛋白中,PR类蛋白的差异表达可能是影响苹果叶片细胞抗病反应的关键因子,研究结果为进一步解析果树抗病分子机制提供了参考。     

不同龄期枝条对苹果轮纹病菌的抗病性研究

本研究采用轮纹病菌分生孢子和菌丝接种不同龄期的苹果枝条,检测枝条皮孔发病率,以明确不同龄期苹果枝条对轮纹病菌的抗病性差异.结果表明,当年生‘富士’苹果枝条的幼嫩部分接种后发病最重,皮孔的发病率达80％以上,随枝条的生长发育,抗病性逐渐增强,枝条发育成熟后,皮孔发病率降至40％左右.在7月份接种的1～5年生‘富士’苹果成熟枝条中,皮孔的发病率没有显著差异.在8月份接种的‘富士’苹果枝条中,1年生枝条的皮孔发病率显著高于5年生枝条的发病率.未发育成熟的苹果枝条对轮纹病菌的抗侵入能力差,枝条发育成熟后抗病能力明显增强.1～5年生枝条上发育完整的皮孔对轮纹病的抗侵染能力没有显著差异,但在枝条的迅速加粗生长期,低龄枝条上皮孔结构变化更大,对轮纹病菌的侵入也更敏感.     

剪口龄期和水分胁迫对轮纹病菌从剪锯口侵染的影响

为了明确剪口龄期和水分胁迫对轮纹病菌从剪锯口侵染的影响,本研究采用脱脂棉蘸取轮纹病菌分生孢子悬浮液接种不同龄期和断水处理的‘富士’苹果枝条剪口,研究了剪口龄期和水分胁迫对轮纹病菌从剪口侵染的影响。结果表明,春季修剪形成的剪口在2个月内对轮纹病菌都较为敏感,接种发病率较高。夏秋季节形成的剪口在2周内对轮纹病菌非常敏感,2周后抗病性逐渐增强。苹果修剪前后,干旱能明显降低剪口对轮纹病菌的抗侵入和抗扩展能力。保护剪锯口不受侵染既能降低苹果苗木带菌率,又能有效控制轮纹病的危害。     

苹果轮纹病菌LAMP快速检测方法的建立

为建立快捷、灵敏检测苹果轮纹病菌Botryosphaeria dothidea的环介导等温扩增(loop-medi‐ated isothermal amplification,LAMP)检测方法,以其内转录间隔区ITS序列为靶标,设计6条LAMP引物,对其特异性进行检测,优化反应条件并建立苹果轮纹病菌的LAMP检测方法。引物特异性检测结果表明,2株苹果轮纹病菌反应结果呈绿色为阳性,而其它3株对照菌株反应结果呈橙色为阴性,表明了LAMP检测引物的高特异性。优化后的LAMP最佳反应条件:温度65℃、扩增时间60 min、FIP/BIP、F3/B3、LF/LB引物终浓度分别为1.0、0.25、0.5μmol/L。LAMP检测方法对苹果轮纹病菌DNA的检测灵敏度达到了100 ag/μL,是常规PCR检测灵敏度的100倍。田间疑似轮纹病组织检测结果发现LAMP方法对苹果轮纹病菌的检出率高达68%,而传统分离鉴定方法的检出率仅为24%。表明所建立的苹果轮纹病菌LAMP快速检测方法简便快捷、特异性好、灵敏度高,尤其适用于基层植保机构对于苹果轮纹病菌的田间快速检测。     

苹果干腐病和轮纹病的发生及防治

苹果干腐病和轮纹病均为害枝干,并为害果实。在苹果主产区,历年因干腐和轮纹烂果遭受损失严重。近年来,随着富士品种栽培面积的扩大,其枝干轮纹病问题日益引起人们的关注。1 病原菌苹果干腐病菌和苹果轮纹病菌都是子囊菌。于腐病菌(Botryosphaeria berengerianade Not.)属于葡萄腔菌属。轮纹病菌(E.berengeriand de Not.f.sp.piricola(Nose)Koganezawa et Sakuma)原属于囊孢壳属。近年来,日本对干腐病菌和轮纹病菌的形态、培养性状和致病性进行了比较研究,于1984年提出轮纹病菌有性阶段的形态结构与干腐     

长期施药果园中的苹果轮纹病菌对戊唑醇和甲基硫菌灵的敏感性

为了监测果园中苹果轮纹病菌对戊唑醇和甲基硫菌灵敏感水平的变化,采用菌丝生长速率法测定了采集自山东烟台地区和北京市昌平区,有较长施药史苹果园中的苹果轮纹病菌对戊唑醇和甲基硫菌灵的敏感性。结果表明:戊唑醇对连续施用戊唑醇5年、每年施药1~2次的果园中苹果轮纹病菌的EC_(50)为0.017 4~0.114 3μg/mL,即该类果园中苹果轮纹病菌对此药的敏感性仍然保持在较高水平,与野生菌株的敏感性非常接近,没有出现敏感性分化的抗药亚群体;甲基硫菌灵对连续施用甲基硫菌灵10年、每年施药2次的果园中苹果轮纹病菌的EC_(50)为0.846 4~4.677 4μg/mL,果园中苹果轮纹病菌与野生菌株相比EC_(50)平均值约上升3.15倍,最低值和最高值分别是已报道敏感性基线的1.19倍和6.59倍,没有出现敏感性发生显著分化的抗药性亚群体。     

苹果属植物寄主应答病原菌胁迫的蛋白质组学研究进展

苹果基因组研究的不断深入,为苹果属植物蛋白质组学研究奠定了基础。近年来,蛋白质组学分析方法在探讨苹果属植物应答病原菌胁迫时,所启动的抗病相关蛋白的差异表达,以及相关抗病机制方面发挥了重要作用。本文针对近年来蛋白质组学研究技术在果树上的应用,综述了苹果属植物应答火疫病、褐斑病、黑星病等主要病害胁迫的蛋白质组学研究进展,分析了蛋白质组学分析技术在苹果属植物相关研究中存在的问题,以期为进一步探究苹果属植物抗病的分子机制提供参考。     

应用GST pull-down技术筛选水稻抗稻瘟病蛋白PID3互作蛋白的研究

 Pid3是从水稻中克隆获得的编码CC-NBS-LRR类蛋白的稻瘟病抗病基因。为了更深入地研究Pid3编码蛋白PID3介导的稻瘟病抗性的分子调节机制,我们应用GST pull-down结合质谱技术,对水稻抗稻瘟病蛋白PID3的互作蛋白进行了鉴定和分析。首先,成功构建了带有GST标签且能够表达PID3的CC-NBS结构域的融合蛋白表达载体pGEX6P1-PID3^CC-NBS,经诱导表达和GST beads纯化后获得GST-PID3^CC-NBS融合蛋白。我们以Pid3的转基因水稻纯合株系Pid3-TP309为研究材料,分别用对其致病和不致病的稻瘟病菌生理小种ZB13和ZHONG-10-8-14接种,然后取叶片分别提取获得两组总蛋白。用纯化后的蛋白GST-PID3 ^CC-NBS分别与两组总蛋白共培养后,利用GST pull-down技术获得了分别在抗病、感病反应中能与PID3^CC-NBS互作的两组候选蛋白。通过质谱技术鉴定这些蛋白的氨基酸序列,比对分析其在抗病、感病途径中与PID3结合的差异性,我们共筛选出31个只在PID3介导的抗稻瘟病反应中能与其特异结合的PID3互作蛋白,这些互作蛋白包括受体激酶、LRR类蛋白、ATP酶、磷酸羧化酶和离子通道蛋白等,广泛参与了调控细胞程序化死亡、胁迫防御反应、物质与能量代谢和信号传导等多个生物学过程。本研究探寻了PID3介导的抗病信号转导过程的关键因子,研究结果为揭示PID3介导的稻瘟病抗病分子机理奠定了良好基础。     

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。     

斑蝥素及去甲斑蝥素对七种植物病原真菌的抑制作用

采用菌丝生长速率法研究斑蝥素和去甲斑蝥素对苹果树腐烂病菌等7种植物病原真菌的抑菌活性.斑蝥素25～200mg/L对7种病原真菌的菌丝生长均有抑制作用,对苹果树腐烂病菌和苹果轮纹病菌EC50分别为0.1 mg/L和8.2 ms/L.去甲斑蝥素50～200 ms/L对苹果树腐烂病菌、苹果轮纹病菌、白菜黑斑病菌、黄瓜菌核病菌的菌丝生长有明显的抑制作用,对苹果树腐烂病菌和苹果轮纹病菌菌丝生长EC50分别为13.6mg/L和80.2mg/L.两种药剂EC卯处理5天后,在扫描电镜中发现两种供试菌菌丝加粗、皱缩,在透射电镜下观察到细胞膜畸形、细胞内空腔增多,出现电子致密物、囊泡等亚细胞结构变化.     

苹果叶片应答斑点落叶病菌胁迫的蛋白质组学分析

In order to elucidate key proteins related to disease-resistance in apple leaves,the total proteins were extracted from control and infected apple leaves, and separated by two-dimensional electrophoresis. The differential expressed proteins were then identified by MALDI-TOF-TOF/MS.In total, 25 differential expressed proteins were detected by Image Master Software. After tryptic digestion, MALDI-TOF-TOF/MS analysis and database searching, 20 protein spots were finally identified, including 11 functional proteins related to photosynthesis, energy metabolism, stress and defense responses. The pathogenesis-related proteins involved in defense responses such as APX, GPX and Mal d1 were differentially expressed in apple leaves. It indicates that these proteins may play a key role in the resistance to A. alternata apple pathotype.     

Comparative proteomic analysis of apple branches susceptible and resistant to ring rot disease

Ring rot disease, caused by the Botryosphaeria berengeriana f. sp. piricola pathogen, is a destructive disease for apple production. To gain further understanding about the defense mechanisms of apple branches against ring rot disease, a comparative proteomic analysis was conducted in our study. We selected two different host responses to B. berengeriana f.sp. piricola infection or challenge, and compared the different proteomes of susceptible and resistant apple branches that had or had not been inoculated with the pathogen. By using 2-DE and MALDI-TOF-TOF MS analysis, 27 differentially expressed proteins were identified in two inoculation assays. According to their function, the proteins were categorized into five classes. In total, according to these two inoculation assays, there were six differentially expressed defense-related proteins identified in the bark of susceptible and resistant hosts, including Mal d1, ASR, and SAMS, which may play key roles for the resistance mechanisms of each host against ring rot disease. We speculated that the only up-regulation of the ASR protein and the dramatic decrease of SAMS in the resistant host may be related to its better disease resistance. In addition, a total of 10 proteins exhibited opposite expression patterns in the bark of susceptible and resistant branches, and they may also be related to the different disease resistances of the two hosts. Due to the complexity of antifungal mechanisms of apple branch hosts against ring rot disease, to obtain more valuable insights about the interaction between the apple host and B. berengeriana f. sp. piricola pathogen, many further investigations will be conducted.     

河南省杨树新生黄叶病害的差异蛋白质组学分析

 采用双向电泳和MALDI-TOF-TOF质谱联用技术,对近年来河南省的新生黄叶病害杨树进行了差异蛋白质组学研究。经图谱分析,在叶片、韧皮部和根部分别获得了98个（61个上调,37个下调）、90个（42个上调,48个下调）和41个（19个上调,22个下调）差异蛋白点。对其中差异表达1.5倍以上的61个蛋白点进行了质谱鉴定,58个蛋白质点鉴定成功,这些蛋白点可归类于26种蛋白质。这些蛋白包括参与光合作用和与抗性相关的蛋白。研究结果有助于揭示病害的发生机理,铁蛋白的鉴定结果对病因的确定具有重要参考价值。     

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。     

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。     

Histological and proteomics analysis of apple defense responses to the development of Colletotrichum gloeosporioides on leaves

Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is an important fungal pathogen known to cause glomerella leaf spot (GLS). The objective of this study was to analyze the infection of C. gloeosporioides on leaves of apple cv. Fuji (resistant) and cv. Gala (susceptible) and apply proteomics techniques to study the apple defense responses 48 h after inoculation (h.a.i.). On both of cultivars, C. gloeosporioides started to germinate at 3 h.a.i. on adaxial surface and produced appressoria adhering to epidermal cell juxtapositions. Histological analysis showed more stratified parenchyma in leaves of cv. Fuji than cv. Gala associated with differences in the chemical composition of cell walls. Total and unique proteins expressed by cvs. Fuji and Gala at 3, 12, 24 and 48 h.a.i. were detected by comparative proteomes analysis. A total of 42 unique proteins expressed at 24 and 48 h.a.i. were identified by MALDI/TOF mass spectrometry, and most of these proteins were identified as directly involved in defense responses.     

Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation

Xylella fastidiosa (Xf) is the bacterial causal agent of Pierce’s disease (PD) as well as other economically important diseases in a number of agronomic, horticultural and ornamental plants. The objective of this research was to tentatively identify proteins that are differentially expressed in grapevines and involved in disease development or defense responses to Xf-inoculation. We comparatively analyzed proteins differentially expressed in Xf-inoculated grape stems using a pair of siblings of 9621-67 (highly susceptible) and 9621-94 (highly resistant) from a cross of Vitis rupestris × Vitis arizonica. Total proteins were extracted from the stems of uninoculated controls and Xf-inoculated plants at 1, 6, and 12 weeks after inoculation, separated by a 2D-PAGE system, and spots representing differentially expressed proteins were analyzed and tentatively identified using LC/MS/MS. Protein identification was performed using BLASTp and tBLASTn against NCBI non-redundant protein databases and EST databases, respectively. Ten tentatively identified proteins were differentially expressed at different time points after inoculation. A thaumatin-like protein and the pathogenesis-related protein 10 from both genotypes, and the 40S ribosomal protein S25 from the susceptible genotype were up-regulated in response to Xf-inoculation. Furthermore, the expression of the thaumatin-like protein increased sharply 12 weeks post-inoculation in the PD-resistant genotype only. Three heat shock proteins, 17.9 kDa class II, protein 18 and 21 were highly expressed in healthy tissues compared with those in tissues infected with Xf, and heat shock protein 21 was not detectable in the Xf-inoculated PD-susceptible genotype. In addition, a down-regulated putative ripening related protein was found in the Xf-inoculated PD-susceptible genotype. Glycoprotein and formate dehydrogenase were identified in the PD-resistant genotype and their expression was constant during plant development. A putative GTP-binding protein was down-regulated in the PD-susceptible genotype. Our results revealed that differential expression of proteins in response to Xf-inoculation was genotype and tissue development stage dependent. The specific roles of these candidate proteins in alleviation or aggravation of this disease are under investigation. The information obtained in this study will aid in the understanding of the mechanisms related to the host–pathogen interactions involved in PD.     

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.