【作者】 徐秋芳；

【导师】 蔡新忠；

【作者基本信息】 浙江大学 ， 植物病理学， 2009， 博士

【摘要】 番茄(Solanum lycopersicum)与叶霉病菌(Cladosporium fulvum)互作符合基因对基因假说，是研究植物与病原互作的模式系统。番茄抗C．fulvum基因(Cf)和叶霉菌的无毒基因(Avr)产物识别后激活下游抗病信号传导，活化各类防卫反应基因的表达，最终表现抗病性。目前对Cf下游信号传导途径的了解还很肤浅。本研究联合采用蛋白质组学分析技术和快速反向遗传学基因功能分析技术——病毒诱导的基因沉默(virus induced gene silencing，VIGS)，筛选和鉴定番茄Cf-4和Cf-9介导的过敏性反应调控基因，获得了以下主要研究结果：(1)建立了一套适于番茄子叶总蛋白分离的双向电泳分析技术体系。通过对双向电泳中几个重要因素，包括上样量、IPG胶条选择、上样方式、聚焦条件等的优化，总结出可以获得高分辨率、高重复性结果的双向电泳操作体系：使用TCA／丙酮沉淀方法提取番茄子叶总蛋白，采用24cm pH3-7NL的IPG胶条，水化上样，300μg的上样量，低电压除盐后8000v 56000～68000vhs进行等电聚焦。(2)开展了番茄抗叶霉病蛋白质组学分析。通过双向电泳分析，分别获取了Cf-4(HR~-)、Avr4／Cf-4(HR~+)、Cf-9(HR~-)及Avr9／Cf-9(HR~+)基因型番茄苗的蛋白质组图谱。对四种基因型番茄的蛋白质组图谱的比较分析结果表明，66个蛋白在Cf-4(HR~-)与Avr4／Cf-4(HR~+)基因型番茄苗中差异表达，其中59个蛋白在Avr4／Cf-4基因型番茄苗(HR~+)中表达量上调，7个下调；71个蛋白在Cf-9(HR~-)与Avr9／Cf-9(HR~+)基因型番茄苗中差异表达，其中59个蛋白在Avr9／Cf-9基因型番茄苗(HR~+)中表达量上调，12个下调。Avr9／Cf-9和Avr4／Cf-4这两个互作中均差异表达的蛋白有43个，其中39个在Avr9／Cf-9(HR+)和Avr4／Cf-4(HR~+)基因型番茄苗中表达上调，4个表达下调。同时分离得到仅在Avr9／Cf-9(HR~+)和仅在Avr4／Cf-4(HR~+)基因型番茄苗中差异表达蛋白，分别为23个和28个。差异表达蛋白点切取后进行MALDI-TOF-MS和MALDI-TOF／TOF-MS质谱分析和数据库检索分析，65个蛋白得到成功鉴定。这些差异表达蛋白功能主要涉及防卫反应、信号传导、代谢、蛋白合成、转录调控和光合作用等方面。(3)采用VIGS技术分析了19个差异表达蛋白对应基因的抗病功能。对差异表达蛋白的序列进行了分析，65个蛋白编码基因中的19个可以在本氏烟或烟草序列数据库中搜索到氨基酸序列一致率68％以上或编码的核酸序列一致率80％以上的同源基因。选取这19个本氏烟或烟草同源基因作为沉默的靶标基因，对其在抗病性中的作用进行了检测分析。采用RT-PCR扩增克隆目的基因片段，并亚克隆至烟草脆裂病毒(Tobacco rattle virus，TRV)基因沉默载体，在本氏烟上进行TRV诱导的VIGS分析，检测沉默植株的抗病性表现。发现蛋白酶体20Sβ亚基、内质网网眼结合蛋白(Luminal-binding protein 5，BiP5)、细胞分化蛋白酶ftsH同源物(Cell divisionprotease ftsH homolog)、促分裂原活化蛋白激酶(Mitogen-activated protein kinase1，MAPK1)、天冬酰胺合成酶(Asparagine synthetase，AS)、ASR4(Abscissic acid，Stress，Ripening)、叶绿体噻唑生物合成蛋白(Chloroplast thiazole biosynthetic protein)等7个基因沉默后植株表现出坏死、矮化、叶片和花器发育畸形等症状。AS、Pip1(phytophthora-inhibited protease 1)、MAPK1、Lemir((?)Lycopersicum (?)sculentum(?)aculin)等4个基因的沉默显著减弱了Cf-4／Avr4和Cf-9／Avr9介导的HR，表明这4个基因对Cf介导的HR起重要调控作用。(4)采用VIGS技术分析了56个ACE(Avr／Cf elicited)基因的抗病功能。对56个ACE基因在本氏烟上进行TRV介导的VIGS分析，其中编码脂转运蛋白、病程相关蛋白、细胞色素P450 76A2、细胞色素P450 72A1、60S核糖体蛋白L10、bZIP转录调控因子BZI-2、激酶互作蛋白及一个未知功能蛋白的8个ACE基因沉默显著减轻了Cf-4／Avr4和Cf-9／Avr9介导的HR，表明这些基因可能在Cf-4和Cf-9介导的HR中起重要调控作用。(5)采用VIGS技术分析了8个泛素介导的蛋白降解途径基因及2个钙离子信号途径基因对Cf介导HR的调控作用。结果表明1个泛素融合降解蛋白(Ubiquitinfusion degradation protein)和2个钙离子结合蛋白(Calcium-binding protein)在Cf-4和Cf-9介导的HR中起作用。综上所述，本研究采用蛋白质组学方法鉴定获取了Cf-4和Cf-9介导HR产生与否差异表达蛋白，并通过VIGS方法分析了蛋白质组水平和转录组水平的差异表达基因在Cf-4／Avr4和Cf-9／Avr9介导的HR中的作用，获取了一批番茄叶霉病菌抗性调控基因。同时，这些研究结果表明结合蛋白质组学分析和VIGS技术分析的体系是筛选和鉴定植物抗病调控基因的有效技术体系，为其它功能基因的筛选和鉴定提供了借鉴。此外，研究结果增进了对番茄叶霉病抗性分子机理的理解，并为番茄叶霉病抗性品种的分子培育提供有利基因资源。更多还原

【Abstract】 The pathosystem of tomato （Solanum lycopersicum） and its leaf mould fungalpathogen Cladosporium fulvum conforms to the gene-for-gene hypothesis and is a modelsystem to study plant and pathogen interactions. Tomato Cf resistance gene productinitiates downstream signal tmnsduction by recognition of C.fulvum avirulence （Avr） geneproduct, resulting in expression of an array of defense related genes, thereby leads tomanifestation of resistance. Little is known about the signal transduction pathway thatdownstream the Cf genes. In this study, proteomic analysis in combination with virusinduced gene silencing （VIGS）, a reverse genetic technology for rapid analysis of genefunction, were employed to screen and identify the genes involved in regulation ofresistance to the tomato leaf mold pathogen. The main results are as follows:（1） A two-dimensional electrophoresis （2-DE） system for separation analysis for totalprotein of tomato cotyledons was established. Through optimization of parameters of2-DE, a manipulation protocol for 2-DE with a high resolution and good reproducibilitywas generated: extract the total protein of tomato cotyledons by the method ofTCA/acetone precipitation, use the 24cm pH3-7NL IPG strips, adopt the rehydrationloading with 300μg protein, 8000v 56000～68000vhs for isoelectric focusingelectrophoresis.（2）. Proteomic analysis for the resistance to C. fulvum was conducted. Proteomicprofiles for cotyledons of tomato seedling carrying Cf-4 （HR）, Avr4/Cf-4 （HR⁺）,Cf-9（HR^-） or Avrg/Cf-9（HR⁺） respectively. 66 spots differently expressed in the Cf-4（HR^-）and the Avr4/Cf-4（HR⁺） seedlings. 59 spots among them are up-regulated, while 7 aredown-regulated in the seedling containing Avr4/Cf-4 showing HR. 71 spots wreedifferently expressed between the Cf-9（HR^-） and Avr9/Cf-9 （HR⁺） seedlings. Among them,59 were up-regulated whereas 12 were down-regulated in Avr9/Cf-9 HR⁺ seedlings. 43spots were differently expressed both between the Cf-4（HR^-） and Avr4/Cf-4 （HR⁺）seedlings and between Cf-9 （HR^-） and Avr9/Cf-9 （HR⁺） seedlings, with 39 up-regulatedand 4 down-regulated in the HR⁺ seedlings. 39 spots were differently expressed onlybetween the Cf-4（HR^-） and Avr4/Cf-4 （HR⁺） seedlings, while 28 were differently expressed only between the Cf-9（HR^-） and Avr9/Cf-9 （HR⁺） seedlings.65 spots were successfully identified by MALDI-TOF-MS andMALDI-TOF/TOF-MS analysis. These proteins belongs to several functional categories,including defense, signal transduction, metabolism, protein synthesis, transcriptregulation and photosynthesis.（3） Function in resistance of 19 differently expressed proteins were analyzed.Nicotiana benthamiana and N. tabacum homologs of the identified 65 differentlyexpressed proteins were selected in these Nicotiana sequence databases, and 19 wereretrieved with sequence identity rate higher than 68% in amino acid level or 80% innucleic acid level. These 19 genes were selected as targets for VIGS analysis in N.benthamiana. Fragments of the 19 N. benthamiana were amplicated by RT-PCR, thencloned into the TRV VIGS vector. The VIGS analysis was performed in N. benthamiana.Silencing of 7 genes, which encode a proteasome 20S beta1.1 subunit, a luminal-bindingprotein 5 precursor （BiP5）, a cell division protease ftsH homolog, mitogen-activatedprotein kinase 1, an asparagine synthetase, ASR4 （（?）bscissic acid, （?）tress, （?）ipening）, and achloroplast thiazole biosynthetic protein, resulted in necrosis, stunt, and abnormal leavesand floral organs in silenced plants. Silencing of genes encodingPIP1（Phytophthora-inhibited protease 1）, mitogen-activated protein kinase 1, asparaginesynthetase and Lemir resulted in a significantly reduced Cf-4/Avr4 andCf-9/Avr9-dependent HR, indicating that these four genes may have essential roles in theCf/Avr dependent HR and disease resistance.（4） 56 ACE （Avr/Cf elicited） genes in Cf-4 and Cf-9-dependent HR were analyzedusing the approach of TRV-induced gene silencing. Silencing of eight genes, whichencode a lipid transfer protein, a pathogenesis-related family protein, cytochrome P45076A2, cytochrome P450 72A1, 60S ribosomal protein L10, bZIP transcription factor BZI-2, akinase interacting protein, and an unkown protein, significantly compromised HR,demonstrating that these 8 genes are important regulators of Cf/Avr-dependent HR.（5）The functions of eight genes related to ubiquitin-mediated protein degrationpathway and two genes related to Ca²⁺ signal transduction were analysed by VIGS. The results showed that one ubiquitin fusion degradation protein and two calcium-bindingproteins play roles in regulation of Cf-4 and Cf-9-dependent HR.Taken together, in this study, proteins differently expressed between seedlingsshowing and not showing Cf-dependent HR were identified through proteomic analysis.The role of genes differently expressed in nucleic acid or protein level in Cf-4/Avr4 andCf-9/Avr9-dependent HR was analysed. 15 genes potentially involved in regulation ofresistance to tomato leaf mould disease were obtained.Results of this study indicate that proteomic analysis in combination with VIGS is aneffective technique system for screening and identification of genes that regulate diseaseresistance. It may be also valuable in screening and identification of other functionalgenes. In addition, the results provide insights into the molecular mechanism of resistanceto C. fulvum, and provide beneficial gene resources for breeding leaf mold-resistanttomato cultivars.更多还原