【作者】 张小利；

【导师】 王晓鸣；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2013， 博士

【摘要】 玉米大斑病（Northern Corn Leaf Blight，NCLB）是由大斑突脐蠕孢（Exserohlium turcicum）引起的叶部病害。本研究进行了玉米抗大斑病种质资源筛选、玉米抗大斑病Ht2基因应用性检测和大斑病菌侵染后玉米的差异蛋白质组学的研究，旨在筛选优良的抗性资源，为玉米抗大斑病机制提供线索。玉米南方锈病（Southern Corn Rust）病原是多堆柄锈菌（Puccinia polysora Underw.），该病害主要流行于热带和亚热带地区。本研究分析了4个玉米抗南方锈病自交系的抗性遗传特征，并定位了自交系冀库12携带的抗南方锈病基因，为更好的利用这些抗病资源提供了理论依据。主要研究内容及结果如下：1.鉴定了499份玉米种质对玉米大斑病菌0、1和N号生理小种的抗性，其中96份对0、1和N号生理小种表现抗（R）以上水平，占到总材料的19.24%；评价了150份种质对玉米大斑病菌0、1、N和123N共4个生理小种的抗性，8份材料对4个生理小种共同表现抗（R）以上水平的，占到总材料的5.33%。2.检测了新报道的5对与玉米抗大斑病Ht2基因连锁的分子标记在11份自交系材料中的扩增情况，包括3对SSR标记，1对SNP标记和1对CAPS标记。结果表明这5对标记在11份材料中都能扩增出条带，但是这些标记缺乏对不同遗传背景下Ht2基因的特异性扩增，即这些标记与Ht2基因不是共分离，标记缺乏普适性，不能用于检测玉米品系中是否携带Ht2基因。3.鉴定了玉米自交系A619Ht2感染大斑病菌13生理小种72h后叶片的差异表达蛋白质。接种叶片与对照比较，137个蛋白质点的表达变化达2倍以上，其中50个蛋白点上调表达，87个下调表达。质谱（MALDI-TOF MS）鉴定了48个蛋白质点，包括6个上调表达蛋白点、10个特异诱导表达蛋白点、20个下调表达点和12个不表达点。这些差异表达蛋白涉及了多种代谢活动，按功能分为9类，其中基础能量代谢类、抗病防御反应类和蛋白质加工存储类分别占到46%、18%和12%。一些重要的抗逆防御相关蛋白上调表达，例如β-葡聚糖酶、SOD、热激蛋白、多胺氧化酶和肽酰脯氨酰顺反式异构酶；而一些参与光合作用和碳水化合物代谢的蛋白质下调表达。4.4个抗玉米南方锈病自交系（冀库6、冀库12、5362和辽2204）分别与感病自交系黄早四杂交，构建了4个F2:3家系。通过田间抗玉米南方锈病的接种鉴定，4个抗病亲本和杂交F1代都表现抗病，F2:3家系出现明显的抗感分离，经鉴定只有冀库12与黄早四杂交的F2:3群体中，抗病家系、分离家系和感病家系分别为27、51和21，符合1:2:1的理论分离比（χ2=1.1633，P=0.5590），初步确定冀库12对玉米南方锈病的抗性是由一对显性基因控制。5.构建了冀库12/黄早四的F2:3家系共328个用于定位冀库12中的抗南方锈病基因，田间抗性鉴定表明冀库12、杂交F1代的抗性为抗或中抗，黄早四表现为高感， F2:3家系抗病家系、分离家系和感病家系分别为77、165和86，符合1:2:1的理论分离比（χ2=0.5061，P=0.7764），再次验证冀库12对玉米南方锈病的抗性是由一对显性基因控制。利用SSR标记和BSA法将抗病基因定位在10号染色体bin10.02，筛选到5对连锁标记，最近的标记phi063与抗病基因的遗传距离是4.2cM。抗病自交系冀库12和齐319的正反交F2代出现了感病株，表明他们所携带的抗病基因不是同一个基因，将冀库12中的抗病基因暂时命名为Rpp12。更多还原

【Abstract】 Northern corn leaf blight (NCLB), caused by the fungus Exserohlium turcicum (Pass.) Leonard etSuggs, is a destructive foliar disease of maize. Identifying and screening the maize resistant germplasmresources to NCLB were conducted to find ideal resistant source. The practicabilities of5molecularmarkers linked to Ht2gene were detected. A comparative proteomic study was conducted to explore themolecular mechanisms underlying the defense responses of the maize resistant line A619Ht2to S.turcica race13. Southern corn rust induced by Puccinia polysora Underw., mainly occurres in tropicaland subtropical area. Four F2:3populations from crossing four resistant inbred lines and susceptible lineHuangzaosi were identified their resistance to southern corn rust and the resistant inheritancecharacteristics were explored. The resistant gene of Jiku12was mapped. Main results of our study weresummarized as follows:1.499maize germplasms inoculated with0,1and N races of S. turcica respectively to identifytheir resistance to NCLB. Among the499germplasms,96germplasms displayed resistant to all thethree tested races and accounted for19.24%of499germplasms.150maize germplasms inoculated with0,1, N and123N races of S. turcica respectively and only8germplasms were resistant to all fore raceswhich accounted for5.33%of499germplasms.2.11maize inbred lines with/without Ht2gene were used by detecting the practicability of5molecular markers linked to Ht2gene, including3SSR markers,1SNP marker and1CAPS marker.The resultes indicated that there was almost none amplified band or sequence which was specific to Ht2gene. Those5markers can not be used for identifying the present of Ht2gene in various maize lines.3. Leaf proteins were extracted from mock and S. turcica race13infected maize resistant lineA619Ht2after inoculated for72h and analyzed for differential protein expression usingtwo-dimensional electrophoresis and mass spectrometry identification. One hundred and thirty-sevenproteins showed reproducible differences in abundance by more than2-fold at least, including50up-regulated proteins and87down-regulated proteins. Forty-eight protein spots were successfullyidentified by MS analysis, which included10unique,6up-regulated,20down-regulated and12disappeared protein spots. These identified proteins were classified into9functional groups andinvolved in multiple functions, particularly in energy metabolism (46%), protein destination and storage(12%), and disease defense (18%). Some defense-related proteins were upregulated such asβ-glucosidase, SOD, polyamines oxidase, HSC70and PPIases; while the expressions ofphotosynthesis-and metabolism-related proteins were downregulated by inoculation with S. turcica.4. Four F2:3segragated populations were constructed by using four inbred lines resistant tosouthern corn rust which were Jiku6, Jiku12,5362and Liao2204as resistance-donor crossed tosusceptible line Huangzaosi. Four inbred lines and all the members of F1population were resistant tosouthern corn rust, while the inbred line Huangzaosi was susceptible. The resistant inheritance analysisindicated that only the segregation ratio of resistance and susceptibility of F2:3populations from Jilu12/Huangzaosi fited a ratio of1:2:1, as indicated by the χ2=1.1633and P=0.5590. The resistanceconditioned by Jiku12was controlled by one dominant gene.5. A328F2:3populations were constructed from Jilu12/Huangzaosi to conduct the gene mappingexperiment. Results of resistance identification furthern showed that the resistance gene of Jiku12wasone dominat gene, since77resistant lines,165segragating lines and86susceptible lines were obserbedand fited the segregation ratio of1:2:1.5SSR markers located on the chromosome bin10.02wereidentified to be linked to resistance gene of Jiku12and the distance of this gene from the nearest markerphi063was4.2cM. According to an allelism test between Jiku12and Qi319, the resistance gene ofJiku12was non-allelic to the RppQ of Qi319. We suggested designating the resistant gene conferred byJiku12as Rpp12temporarily.更多还原