【作者】 陈雅平；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 小麦白粉病是由白粉病菌(Erysiphe graminis Dc.f.sp tritic Marchal)引起的世界性真菌病害，培育抗性品种是控制白粉病的有效途径。上世纪八、九十年代我国原有的抗白粉病小麦品种大部分都含有Pm8抗源，但是随着白粉菌生理小种的不断变化，Pm8已经逐渐失去了抗性，寻找和利用新的白粉病抗性基因对于我国小麦生产有重要意义。南京农业大学细胞遗传所培育的6VS／6AL易位系带有的抗白粉病基因Pm21抗性强、抗谱广，因此对Pm21基因及白粉病抗性相关基因进行克隆具有重要的意义。 构建cDNA文库是基因克隆的基础，但是由于小麦基因组庞大，所需文库含克隆数太多，后续筛选工作较为复杂，所以本研究以Pm21基因供体物种簇毛麦(2n=14，VV)为材料构建了白粉菌诱导的簇毛麦叶片cDNA文库。此文库包含30万个重组克隆，平均插入片段为1.6kb，用本研究克隆到的谷胱甘肽还原酶(Ta-GR)基因，蓝铜结合蛋白基因(BCB)和谷胱甘肽硫转移酶基因(GST)等作探针进行杂交筛库，杂交信号明显，重复性好。 根据抗病基因保守结构域设计简并引物以及以数据库中已经定位的小麦ESTs序列为基础设计特异引物，以经白粉菌诱导后的抗病易位系和感病扬麦5号cDNA为模板进行RT-PCR筛选。共获得4个与小麦白粉病抗性相关的侯选基因。其中，利用特异引物，从易位系中分离获得了两个cDNA片段(Ta-RPM,Ta-M1a)，它们的核苷酸序列分别与大麦NBS-LRR类抗病蛋白(AAB96982.1)及大麦CC-NBS-LRR抗病蛋白MLA13基因(AF523682.1)序列同源性为64％和87％。利用简并引物从易位系cDNA中扩增获得两个cDNA序列(Th8，Th12)，推测TH8为小麦蛋白激酶基因片段，TH12为小麦单脱氢抗坏血酸还原酶基因片段，利用中国春缺体四体将TH8和TH12分别定位于小麦5D和3D染色体上。并对上述分离的基因进行了序列比较研究和蛋白一级、二级结构比较分析。 应用抑制性消减杂交(SSH)技术，以Pm21受体亲本扬麦5号及与其回交7代的小麦／簇毛麦6VS／6AL易位系为材料，建立白粉菌诱导的叶片SSH文库，获得500个阳性克隆，对其中部分克隆测序，并与GenBank中的序列进行BLAST分析，获得12个与抗病相关的EST序列。本研究详细地分析了其中三个EST(Ta-APX,Ta-LRR2，HvGR)。 通过电子克隆方法首次克隆到了全长小麦抗坏血酸过氧化物酶基因(Ta-APX)，并根据全长序列设计特异引物，利用中国春缺体四体基因组DNA为模板进行PCR扩增，将Ta-APX定位于小麦第七部分同源群7A和7D染色体上。构建表达载体pET32(a)更多还原

【Abstract】 Powdery mildew, caused by Erysiphe graminis DC. f.sp.tritici Marchal, is one of the major diseases of common wheat worldwide. Development and utilization of wheat cultivars with resistance gene is an efficient, economic and environmentally safe strategy to control powdery mildew. Many powdery mildew resistance genes had already lost their resistance effect because of variation of prevalent races. Pm21, originally introduced into wheat from H. villosa through chromosome translocation of 6VS/6AL, is an effective powdery mildew resistance gene with broad spectrum in the world and has a good promise in wheat breeding. So cloning of Pm21 gene and powdery mildew resistance-related genes will play an important role in future disease resistance breeding.Construction of cDNA library is a prerequisite for gene cloning. But it is difficult to construct and screen a cDNA library from common wheat because of its large genome. In this study, a cDNA library was constructed using cDNA from leaves of diploid Haynaldia villosa (2n=14, VV) inoculated with Erysiphe graminis. The cloning vector was an expression plasmid vector pSPORT1 which was introduced into E.coli DH10B. The cDNA library contains about 30,0000 recombinant cDNA clones, with average insert size of 1.6kb and is successfully screened with the probes of wheat glutathione reductase gene (Ta-GR), a blue copper-binding protein (BCB) gene, a glutathione-S-transferase (GST) gene and other genes cloned by ourselves.RT-PCR screening approach using degenerate primers designed based on conserved domains of plant resistance disease genes and specific primers designed according to the ESTs which had been mapped onto wheat homologous group 6 was applied to isolate resistant related gene. Ta-RPM and Ta-Mla isolated from translocation line with specific primers are high homologous to barley NBS-LRR resistant protein b7 (AAB96982.1) and barley CC-NBS-LRR resistance protein MLA13 (AF523682.1) , respectively. TH8 and TH12 obtained by degenerate primers have high homology to Arabidopsis Ser/thr protein kinase and human MDHC gene, respectively. Southern blot with Chinese Spring nullisomic/tetrasomic lines was used to assign the genes of TH8 and TH12 to chromosome 5D and 3D, respectively. The nucleotide and deduced amino acid sequence of the four clones were analyzed and characterized.更多还原