【作者】 黄秀丽；

【导师】 陈捷；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 弯孢菌叶斑病(Curvularia lunata(Wakker)Boed)是威胁我国玉米可持续生产的主要病害之一,培育和种植抗病品种是防治该病害发生与危害的根本途径。然而,目前我国缺少对该病免疫的品种,且病原菌具有明显的致病性分化现象,极易导致品种抗性丧失,为此,从目前已有的抗性种质资源中挖掘抗病基因,对培育持续抗性新种质具有重要意义。本研究利用以双向电泳(2-DE)和质谱(MS)鉴定为核心的蛋白质组学技术,筛选玉米弯孢菌叶斑病抗性相关蛋白质和基因,并研究其在寄主抗性中的作用,主要内容及结果如下:1.分析了弯孢叶斑病菌侵染玉米抗、感自交系后防御相关因子变化规律。结果表明:病原菌侵染后,抗性自交系中SA及O2-含量显著提高,病程相关蛋白PR-1、PR-10、PAL、CAT、POX等的转录水平也显著提高,而感病自交系中ABA含量显著提高,防卫基因转录水平也有提高但是不显著。由此推测,SA增强了玉米对弯孢菌叶斑病的抗性,ABA则相反,增强了玉米对该病害的易感性,而ROS的积累在早期抗性反应中发挥重要作用,PR-1、PR-10、PAL及ROS清除酶类也参与了玉米抗弯孢叶斑病菌侵染的防御反应。2.建立了基于2-DE技术的玉米叶片蛋白质高效分离系统。通过对蛋白质样品制备、蛋白质裂解液配方、IPG胶条pH范围和等电聚焦电泳时间等参数的优化,建立了玉米叶片总蛋白质分离的2-DE体系。同时,建立了以去除叶片高丰度蛋白Rubisco为主要目标的Mg/NP-40及PEG预分离体系,明显提高了2-DE系统对低丰度蛋白质的分离效率。3.鉴定出了部分与玉米抗弯孢菌叶斑病相关的蛋白质。通过对抗、感玉米自交系接种弯孢叶斑病菌后叶片蛋白质2-DE图谱的比较,利用MALDI-TOF MS/MS成功鉴定了36个差异表达的蛋白质点,其中与该病抗性密切相关的蛋白质包括22 kDa干旱诱导蛋白、脱落酸/成熟诱导蛋白Asr、萌发素类似蛋白GLP、谷胱甘肽过氧化物酶GPX、抗坏血酸过氧化物酶APX、翻译起始因子eIF-5A和ras相关蛋白等。光合作用、能量代谢等相关蛋白质也参与了玉米抗弯孢叶斑病菌侵染应答反应。综合分析,玉米耐受干旱和抗氧化胁迫相关蛋白质可能是玉米抗弯孢叶斑病菌侵染的重要因子,在玉米耐受逆境胁迫及抗病反应的“交叉对话”中发挥重要作用。4.克隆了玉米弯孢菌叶斑病抗性相关基因ZmDip。根据质谱鉴定结果,成功克隆了玉米22 kDa干旱诱导蛋白编码基因ZmDip且实现了其在大肠杆菌E. coli中的高效表达,并纯化了目的蛋白,为制备该蛋白的多克隆抗体提供了条件。本研究还发现ZmDip与Riccardi和Jeanneau等报道的ZmAsr1基因有98%的一致性,蛋白序列有99%的一致性,且均定位于10号染色体上,由此推测,ZmDip与ZmAsr1为同一个基因,两者之间核苷酸及编码蛋白的微小差异可能与基因来源即玉米基因型有关。5. ZmDip表达特性及弯孢叶斑病菌侵染后转录水平变化分析。利用半定量RT-PCR对ZmDip的表达特性进行了分析,发现该基因在正常生长的玉米根部表达,叶鞘不表达,而叶片中该基因的表达比较复杂,可能与玉米基因型有关。外源ABA、SA均能诱导该基因的表达,弯孢叶斑病菌侵染后,抗、感自交系中该基因的转录水平也发生变化,但与接种后寄主植物叶片中ABA、SA含量变化没有一致性,由此认为,ZmDip基因的表达可能主要受ABA、SA以外的其它路径调控。6.ZmDip表达提高了玉米叶片抗弯孢叶斑病菌侵染的能力。ZmDip基因在玉米离体叶片中的表达,促进了ROS主要是O2-的积累,抑制了弯孢叶斑病菌的侵染。因此,初步认为ZmDip基因表达调控了ROS代谢,从而提高了玉米对弯孢叶斑病菌侵染的抵抗力。至于该基因参与寄主植物抗性反应的具体路径,还需深入研究。更多还原

【Abstract】 Maize leaf spot caused by Curvularia lunata (Wakker) Boed is one of the most widely distributed diseases in maize growing area of China. The disease ever outbroke seriously and resulted in a great grain loss in 1996 in northern China. In the long run, breeding resistant cultivars is the most economic and effective measures to control this disease. However, until now few cultivars possess high resistance to the disease. Furthermore, the resistance of cultivars may be easily lost due to the frequent genetic variance of the pathogen. Thus exploring proteins or genes related to the host resistance from tropic germplasm appears to be the most pressing task. Here, a proteomic approach mainly including 2-dimensional gel electrophoresis (2-DE) and MALDI-TOF MS/MS techniques was applied to screen and identify defense-related proteins from resistant inbred lines widely planted in China, then genes encoding corresponding proteins were cloned, eventually their functions in host resistance were discussed. The main results were as follows:1. Changes of defense-related factors in maize response to C. lunata infection. After inoculation with C. lunata, the content of SA and ROS especially O2- was significantly increased in resistant inbred lines. Besides, the transcription levels of defense genes including pathogenesis-related genes PR-1, PR-10, PAL, CAT, and POX were also significantly enhanced. While the content of ABA was found markedly increased in susceptible ones, and the transcription levels of defense genes were also increased to some extent but not significantly. Based on the results, SA was speculated to be positively correlated with maize resistance to C. lunata attack, but ABA was on the contrary. The accumulation of ROS in the early stage of pathogen infection was detected and inferred to stimulate the enhancement of maize disease resistance. Meanwhile PR-1, PR-10, PAL and ROS scavenging enzyme also played important roles in maize resistance to C. lunata infection.2. Optimized 2-DE system to separate proteins from maize leaves. A 2-DE system to effectively extract and separate total maize leaf proteins was established by optimizing protein extracting methods, sample lysis buffer, pH value of IPG gel and time for IEF. Meanwhile, Mg/NP-40 with PEG fraction was evidenced to be an effective method to remove high abundant proteins mainly Rubisco from total leaf protein samples according to Kim’s methods (2001). The capacity of 2-DE system to visualize and detect low abundant proteins improved greatly. 3. Identification of proteins associated with resistance to Curvularia leaf spot of maize. After inoculation with C. lunata, proteomic profiles of maize leaves between resistant and susceptible inbred lines were analyzed. Thirty-six differentially expressed protein spots resolved on the 2-DE gels were successfully identified by MALDI-TOF MS/MS. The results showed that the proteins were associated with photosynthesis, respiration, drought and oxidative stress tolerance as well as signal transduction. Of those stress related proteins, 22kDa drought-inducible protein, ABA/stress/ripening inducible protein (Asr), germin-like protein (GLP), glutathione peroxidase (GPX), ascorbate peroxidase (APX), translation initiation factor (eIF-5A) and ras related protein were unique or up-regulated in the resistant inbred lines and supposed to be implicated in host defense response to C. lunata infection. Meanwhile, drought and oxidative stress-related proteins were primarily considered to be most closely involved in maize resistance to pathogen infection. Based on the above and other observations, it was deduced that there might be cross-talk between host resistance to abiotic and biotic stresses.4. Cloning of defense-related gene ZmDip and its expression. Accoding to mass spectrometry result,the gene encoding 22 kDa drought inducible protein in maize was successfully cloned through RT-PCR and named as ZmDip. To carry out gene function analysis, the high level expression of ZmDip in E. coli was achieved, and subsequently purified for antibody preparation. Besides, ZmDip (Genbank accession No. EU164846) showed 98% and 99% consistency in gene and protein sequence with that of ZmAsr1 ( AX297905 , NCBI ) , respectively, so they were supposed to be one gene even though the minor difference existed, which was perhaps partly attributed to genotypes of maize inbred lines.5. Analysis of transcription levels of ZmDip in normal and inoculated conditions. The expression patterns of ZmDip in different tissues of maize were analyzed with semi-quantitative RT-PCR. The gene was constitutively expressed in root but not in leaf sheath. Its expression was very complicated in leaves, and depended probably on genotypes of maize inbred lines. Transient expression of ZmDip in maize leaves was induced when treated with ABA and SA. Moreover, differential transcript levels of ZmDip was found in resistant or susceptible inbred lines after inoculated with C. lunata, but no clear trend was found regarding to their correlation with maize resistance levels and content of ABA and SA in inoculated host. Thus it was deduced that ZmDip might be mainly regulated by other singal pathways besides ABA and SA.6. Expression of ZmDip improved host resistance to C. lunata infection. To discover the roles of ZmDip played in maize resistance to C. lunata infection, the recombinant vector pBI-ZmDip-Gus was constructed. ZmDip successfully expressed in leaves in vitro (Huangzao 4) through Agrobacterium-mediated transformation. The expression of ZmDip led to the accumulation of ROS specially O2- and remarkably reduced leaf spot area in transformed leaf tissue by subsequent challenge inoculation as compared with control. Taking together, ZmDip played a positive role in strengthening maize resistance to C. lunata infection, but the exact role ZmDip played needed to be further discussed.更多还原