【作者】 李文凤；

【导师】 吴立人；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2001， 博士

【摘要】 小麦条锈病是一种世界性病害，严重威胁着小麦生产，目前尚未发现成功克隆小麦抗锈基因的文献报道。小麦抗条锈病基因Yr10可抵抗我国目前出现的所有条锈菌生理小种，而且在生产上还基本没有被利用。因此，本文以Yr10的载体品种Moro作材料，首次应用差异显示RT-PCR技术，从小麦中分离了小麦与条锈菌抗性反应过程中相关基因的cDNA片段；并首次以小麦抗条锈病基因Yr10的近等基因系为材料，运用植物抗病基因保守序列策略，探索了分离小麦抗条锈病基因Yr10的候选基因的新途径，取得了以下重要进展： 以小麦条锈菌（Puccinia striiformis West.）菌系75078和CY27分别接种含抗条锈病基因Yr10的小麦品种Moro，组成小麦与条锈菌亲和及不亲和互作组合。采用差异显示RT-PCR 使用3个锚定引物T₁₁MN（M为A、G、C简并碱基，N为A、G或C），与40条随机10聚体引物组合，分析上述处理及其与不接种对照间mRNA水平的差异。发现不同引物组合间的扩增结果差别很大，有的能扩增出很丰富的条带，有的只扩增出稀疏的条带，而且不同引物组合扩增产物间的多态性不同。 对接种后24、48、72和96小时样品的分析，共回收到146条差异带，其中接种后24和48小时样品回收到78条差异带。 进一步将这78个再扩增产物分别与其对应的总cDNA探针进行反向Northern杂交，获得1个在不亲和组合和亲和组合间均表达，而在不接种对照中不表达的片段，将这片段命名为DD-1。将DD-1克隆到T-easy载体上，进行序列测定。DD-1长度为316bp，在GenBank中的注册号为AY015491。同源性检索没有发现与其明显相似的已知序列，推测DD-1为一个新基因片段。 Northern点杂交显示DD-1片段在亲和与不亲和组合中都有表达，但在不亲和组合中表达量较大。接种72小时后，DD-1在亲和组合中表达量减少，而在不亲和组合中表达量无明显变化。 以Yr10的近等基因系作材料，根据多数已克隆R基因都具有的NBS区设计一对简并引物，用Touch down PCR程序扩增它们的cDNA，结果在Yr10/6×Avocet S中稳定出现一条约800bp的条带，而在其背景材料Avocet S中没有这条带。序列分析表明该cDNA片段长度为771bp。核苷酸序列同源性检索发现， 中巳农业科学院傅土学位论文GenBank中有小麦抗条锈基因YrIO的序列（2000年12月登记），本实验获得的 cDNA片段与该 Yr！0基因高度同源（99％），只在 foo mRNA的第 613位的T（差异片段为G）、1006位的G（差异片段为A）和 1062位的G（差异片段为A）不同。氨基酸序列同源性检索发现，该片段编码氨基酸序列与Yrlo蛋白的203－459位氨基酸序列有99％的同源性，只在205位的L（差异片段编码氨基酸为V）不同。此外，该片段编码氨基酸序列还与多个R基因编码蛋白有不同程度的相似性u刁2们。 简易法提取Yr 0／6 x Avocet S其背景材料Avocet S的基因组DNA，根据差异片段序列结果设计一对特异引物，同样用 TOuch down PCR程序进行扩增，得到一条在Yrlo历XAvocet S中稳定出现的约工刀kb的条带，而在其背景材料Av。cet S中没有这条带出现。推测该基因含有一个长度大于 1．okb的内含子。 利用已有资源分别扩增 77fop CDNA片段对应基因的 5’部分和 3’部分。用分别对应于 771hp CDNA片段 ITlltThA序列的 5’端第 12〕0位，和 YrIO基因的第卜 18位的核昔酸序列合成一对特异引物，扩增 771 hp CDNA片段对应基因的 5’部分；用对应于 77fop。DNA片段 mRNA序列第 752J 位，和 Yr10基因的第36113630位的核苦酸序列合成一对特异引物，扩增77fop CDNA片段对应基因的 3’部分。以上述两对引物分别扩增 Yrlo／6XAvocet S总 cDNA和基因组DNA，均得到特异扩增产物，大小与预计产物长度相符合。每对引物从总CDNA和基因组DNA扩增得到的片段大小都相同。 对上述 5’和 3’部分扩增产物进行克隆和测序，加上 771 hp片段的序列，得出 77fop cDNA对应基因的 2475nt全长 InRNA编码区序列，含一个编码 82’个氨基酸的ORF，其编码蛋白的一级结构具有LZ－NBS七RR结构特征。该mRNA对应基因中含有一个长度大于1．okb的内含子。用地高辛标记上述从Yrlo伍XAvocet v总。DNA中扩增的 3’部分 111 sbp片段作为探针，与 ech和 Bdl完全消化基因组DNA杂交，表明该基因是单拷贝的。 本实验用于扩增1118hP DNA片段的引物只与 INO基因特异结合，该片段可以作为yrlo基因理想的分子标记。更多还原

【Abstract】 AbstractStripe rust (yellow rust), caused by Puccinia striiforms f. sp. tritici West., is one of the most important diseases on wheat throughout the world. Now we haven’t found the cloning of wheat rust disease resistance gene. Wheat stripe rust disease resistance gene YrlO confer resistance to all the races in China. So we used wheat cultivar Moro(containing YrlO gene) and NIL of YrlO as materials to clone the resistance-related cDNA fragments and candidate gene for YrlO.Using the total RNA extracted from seedlings of wheat cultivar Moro (YrlO) inoculated with the avirulent isolate CY27 and the virulent 75078 of Puccinia striiformis West, respectively, we optimized silver-stained mRNA differential display RT-PCR to get a plentiful and high-resolution silver PAGE. Combining three anchored primer T11MN (M is degenerate of A, G, C; N is A, G or C) with 40 10-mer arbitrary primer, differences on mRNA level were analyzed among compatible interaction, incompatible interaction and not inoculated control. Some primer combinations amplified plentiful bands, while others combinations amplified only a few bands.A total of 146 polymorphic bands were reamplified from the samples collected 24, 48, 72, 96h after inoculation. A cDNA fragment DD-1 was cloned with T-easy vector after reverse Northern blotting of 78 differential displayed bands from the samples of 24 and 48h and sequencing showed that it was 316bp (GenBank accession number AYO15491). It has no obvious homologous sequences in GenBank.Northern dot blot hybridization analysis using probe of DD-1 as the probe uncovered that DD-1 was induced by both isolates, but the transcript abundance was less in the compatible interaction than in the incompatible interaction. Seventy-two hours after inoculation, the transcript abundance of DD-1 had no obviously change in the incompatible interaction though it decreased in the compatible interaction.Degenerated primers were designed according to the conserved NBS domain of R genes. Amplification was done with Touch Down PCR using the cDNAs astemplates reverse transcribed from YrlO NILs mRNAs. A 771bp PCR product appeared in the resistant Fir/0/6 X Avocet S but not in the susceptible background parent Avocet S. It has a high homology (99%) with YrlO gene(accession in Dec. 2000) in both nucleotide and amino acid sequences.With primers designed according to the sequence of the DNA fragment, an about 2.0kb fragment was amplified from the genomic DNA of YrlO/6 X Avocet S. It showed that this gene has an about 1.0kb intron.To get the 5’-terminal and the 3’-terminal fragments, a pair of primers was synthesized according to 12-30 of the 771bp cDNA and 1-18 of YrlO mRNA and a pair of primers according to 752-771 of 771bp cDNA and 3611-3630 of YrlO mRNA. A 636bp fragment at the 5’-terminal and an 1118bp fragment at the 3’-terminal were amplified by PCR with the primers from the cDNA of YrlO/6X Avocet S. The same fragments were also amplified from the genomic DNA.The full sequence of the mRNA was gained by combining the 3 cDNA fragments. The full mRNA with 2475 nucleotides has an ORF containing 824 amino acids. The deduced protein primary structure has LZ-NBS-LRR domain. Southern blotting using the genomic DNA digested with EcoR I and BamH I and the DIG-labeled 1118bp probe showed that the obtained gene is of single copy.The primers using to amplify 1118bp fragment at the 3’-terminal binds to YrlO gene specifically. The 1118bp fragment is a good molecular marker of wheat stripe rust disease resistance gene YrlO.更多还原