【作者】 王晓清；

【导师】 王国英；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 植物抗病机制一直是植物病理学研究的热点。近些年来,人们对植物应答病原菌胁迫的作用分子及其作用方式已有了较深入的认识,但抗病途径的一些节点仍不清楚。抗病突变体是研究植物抗病机制的有效工具,用蛋白质组学方法对这些突变体进行研究,可以较全面地揭示出受突变基因调控的蛋白及其介导的抗病相关途径。CPR30是植物抗病反应的一个负调控因子,拟南芥功能缺失突变体cpr30具有自发的超敏反应和系统获得性抗性现象,是典型的获得性抗性突变体。本文用蛋白质组学方法对cpr30突变体中的差异表达蛋白进行了研究并初步分析了CPR30对抗病途径的调控机制。本文用双向电泳对野生型和cpr30的总蛋白进行了分离,凝胶图谱分析结果显示cpr30的双向电泳图谱中有52个蛋白点的表达量发生了显著变化。将这些差异表达的蛋白点经酶解后用MALDI-TOF-MS进行质谱鉴定,数据分析结果表明有33个蛋白点得到鉴定。这些差异表达蛋白按生物学功能可分为以下5类：防御相关蛋白；氧化还原相关蛋白；能量代谢相关蛋白；信号转导相关蛋白；蛋白折叠及动态平衡相关蛋白。用荧光定量PCR对部分编码差异表达蛋白的基因进行了转录水平分析,结果表明其转录水平和翻译水平并不完全一致,这可能与蛋白表达的滞后性和翻译后修饰有关。对部分差异蛋白进行了Western blot验证,结果表明其蛋白表达量的变化与蛋白质组学的实验结果是一致的。本实验室前期研究发现pad4cpr30双突变体几乎完全抑制了cpr30的矮小表型,并且阻断了cpr30中一些抗病因子的表达。本文对pad4cpr30双突变体进行了蛋白质组学研究。双向凝胶图谱分析结果显示共有9个蛋白点的表达量发生了显著变化,质谱结果显示这9个蛋白点都得到了可信的鉴定结果。功能分析表明这些差异蛋白主要参与蛋白折叠、细胞氧化还原动态平衡、能量代谢和氨基酸代谢等四个途径。其中有6个蛋白在cpr30中的表达也发生了显著变化,因此这6个蛋白可能也参与CPR30调控的抗病途径。由于CPR30是一个F-box蛋白,可能通过与SKP1和Cde53形成泛素连接酶复合体来参与泛素-蛋白酶体降解途径。因此cpr30中表达发生上调的蛋白有可能是CPR30作用的底物。通过酵母双杂交实验对CPR30的互作蛋白进行了筛选,结果表明只有SGTla及其同源蛋白SGT1b能与CPR30相互作用。进一步通过构建sgt1a cpr30和sgtlb cpr30双突变体来研究SGTla和SGTlb在cpr30中的作用。表型观察和台盼蓝染色结果表明这两个双突变体部分恢复了cpr30的株高并抑制了其细胞坏死现象。抗病性鉴定结果表明双突变体的感病情况介于野生型和单突变体之间,即部分恢复了cpr30的感病表型。这说明SGTla和SGTlb参与了CPR30介导的抗病途径,并在该途径中起正调控作用。更多还原

【Abstract】 Much attention has always been paid to the molecular mechanisms of plant defense response against pathogen infection. Over the years, many new components involved in plant immunity have been elucidated, yet some joint points of the entire pathway of plant defense are still unclear. The mutants that exhibit native disease resistance are powerful materials to dissect the plant defense mechanisms. Furthermore, proteomic study on these materials can help us obtain systematical cognitions for protein expression profiles of the defense response involved in these mutants. The F-box protein CPR30is a negative regulator of plant defense response, and its loss-of-function mutant cpr30, which show HR-like lesion formation and SAR-like constitutive defense responses, is a typical gain-of-resistance mutant. In the present study, we analyzed the proteome of cpr30and explored its defense mechanism preliminarily.The total proteins of cpr30and the control (wild type) were separated by2-D electrophoresis, and analysis of the2-DE patterns revealed that the expression level of52proteins had been changed significantly in cpr30. Among the52spots digested with trypsin and identified by MALDI-TOF mass spectrometry,33spots, a major of which were strongly up-regulated and had sufficient abundance, obtained high and credible scores. The identified proteins were classified into five groups based on their putative functions, including pathogen-defense, redox homeostasis, energy metabolism, signal transduction, and protein folding and homeostasis. We compared the mRNA abundance of some genes encoding affected-proteins via quantitative RT-PCR to see if there are any changes between mRNA level and protein level, and the results showed that the protein level was not coincided with the mRNA level, which might be due to the posttranslational modification. We further confirmed the expression pattern of some important proteins via Western blot. Consistent with our2-DE results, Western blot analysis also indicated that the expressions of these proteins were indeed greatly affected in cpr30. In addition, the Western blot results also demonstrated that the2-DE analysis yielded fairly accurate measurement of protein expression.We also studied the proteome of the double mutant pad4cpr30, which almost completely suppressed the phenotype of cpr30. Nine protein spots whose abundance had changed significantly were identified by2-DE coupled with MALDI-TOF-MS. Function analysis indicated that these proteins were involved in protein folding, redox homeostasis, energy metabolism and amino acid metabolism. Six spots of which increased significantly in pad4cpr30were also increased in cpr30, indicating the possibility that they may involved in the defense pathway regulated by CPR30.The F-box protein CPR30can interact with ASK proteins to form SCF complex, so it might degrade some yet unknown substrates through ubiquitination, and the substrates might be one or more proteins that induced in cpr30. The yeast two-hybrid assays were performed to screen the substrates interacted with CPR30. The results showed that only SGTla and its homolog SGTlb could interact with CPR30. The function of SGT1a and SGTlb involved in cpr30were explored through the generation of the double mutants sgtla cpr30and sgtlb cpr30. The appearance of these double mutants indicated that sgtla and sgtlb partially suppressed the dwarf morphology of cpr30, as well as cell death and disease resistance conformed by typan blue stain and pathogen infection. Above all, these results suggested that SGTla and SGTlb played positive roles in the defense response regulated by CPR30.更多还原