【作者】 闫红飞；

【导师】 刘大群； 杨文香；

【作者基本信息】 河北农业大学 ， 植物病理学， 2003， 硕士

【摘要】 小麦叶锈病是一种世界性病害，严重威胁小麦生产。迄今为止，所克隆的抗病基因中关于小麦抗叶锈病基因的成功报道较少。目前世界上还没有对小麦Lr38单基因系具有毒性小麦叶锈菌株，其致病型最高为2。本文应用Lr38和致病型分别为0和2的99-8-9-2-1（0）及99-5-30-2（2）两个叶锈菌菌株为材料，应用差异显示技术，分离叶锈菌与小麦互作反应中诱导表达的mRNA，并将所得mRNA反转录成cDNA。然后进行PCR扩增比较不同处理间出现的差异表达的cDNA片段并对其进行进一步分析，取得了一定的进展。 小麦叶锈菌（Puccinia recondita f.sp.tritici）两个菌株99-8-9-2-1（0）及99-5-30-2（2）分别接种含Lr38的单基因系材料，组成强不亲和及弱不亲和互作组合，应用差异显示RT-PCR技术，使用分别带有HindⅢ酶切位点的3个锚定引物AAGCT₁₀N（N为A、G或者C），与24条10mer随机引物组合，分析以上处理和不接菌对照处理的mRNA表达水平的差异。发现不同引物组合间扩增结果差别较大，且不同引物组合扩增产物间多态性也不同。 对接种后12，24，36，48，60和72h的小麦样品分别取样，提取叶片组织总RNA以代替mRNA进行分析，结果发现在24h即有差异条带出现，但数量很少。在36h出现差异条带明显增多，表现出病原物与寄主植物互作而诱导寄主组织抗病基因的表达。将获得的差异条带回收，并在对应的基本相同条件下扩增以便进行后继工作如Northern杂交检测、cDNA文库构建、克隆、测序等。更多还原

【Abstract】 Wheat leaf rust disease, caused by Puccinia recondita f. sp. tritici, is one of the most important diseases on wheat production throughout all the world. Now we haven’t found the cloning about wheat leaf rust resistance gene in cloned, and no wheat leaf rust races have been found to overcome resistance of Lr38. So Lr38 and two leaf rust races 99-5-30-2 which express type is 2 and 99-8-9-2-1 which express type is 0 as the materials of this research, isolated the total RNA of wheat leaf which inoculated by two leaf rust races and control, reverse tanscription the mRNA to complement DNA, then compare the bands of two test and control by DDRT-PCR methods. At last we make a break through in this research.Two races of leaf rust inoculated the Lr38 and no inoculated as the control. Combining three kinds of anchored primer T10N(N is A, G or C), which is containing a HindIII recongnition site, and with 24 kinds of 10 mer arbitrary primer, difference on mRNA level were analyzed among between the two tests and control by DDRT-PCR. We found the difference between differential primers comebination is big, and the The difference between differential primer combination is big, and the amplification products polymorphism is also difference.The differential time 12, 24, 36, 48, 60 and 72 hours of wheat leaf samples which is inoculated by two leaf rust races and control. We use the extracted total RNA of wheat leaves to replace the mRNA analysis the difference of resistance gene induced expression. We start to find the differential cDNA bands at 24 hours after inoculated, but the quantity is small. The high level resistance gene expression after 36 hours. Restrive the differential cDNA fragment from the PAG and reamplified it at the same parasites which can make us do the others research works, for example Northernblotting, molecular hybrid, cloning, sequence checking and so on.更多还原