【作者】 于玲；

【导师】 陈佩度；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2001， 博士

【摘要】 小麦白粉病是由白粉菌（Erysiphe graminis Dc. f. sp tritic Marchal）引起的世界性真菌病害，控制白粉病的有效途径是培育抗性品种。南京农业大学细胞遗传所培育的6VS/6AL易位系带有目前我国最有效的抗白粉病基因Pm21。本研究以抗病易位系6VS/6AL及感病亲本扬五为材料，分离、克隆、分析与白粉病抗性相关的基因，以期深入研究Pm21基因的抗病分子机制，并为最终克隆Pm21基因奠定基础。 1．根据抗病基因保守结构域设计简并引物，并参照大麦、小麦、水稻等作物中分离的防卫反应基因及大麦Mlo基因设计特异引物，以经白粉菌诱导后的易位系和扬麦5号cDNA为模板进行RT-PCR筛选。共获得8个与小麦白粉病抗性相关的侯选基因。其中，利用特异引物，从小麦中分离获得了与大麦病程相关蛋白1、类甜蛋白及小麦β-（1.3，1.4）葡聚糖苷酶高度同源的基因序列，进一步利用RACE技术获得了小麦病程相关蛋白1全长cDNA克隆（Ta-Prl）（GenBank登录号AF384143），通过筛选已构建的易位系cDNA文库，获得了小麦类甜蛋白全长cDNA克隆（Ta-TLP）（GenBank登录号AF384146）；利用简并引物，从小麦易位系cDNA中，首次分离Cyclophilin基因（Ta-Cyp）（GenBank登录号AF384147）和H~+-ATP酶基因序列（Ta-MAH）（GenBank登录号AF384148）以及与大麦、玉米等植物NBS-LRR抗病蛋白有极高同源性的cDNA序列（Ta-RP）；根据大麦白粉病基因Mlo，设计二对特异引物。将易位系经白粉菌诱导后，提取mRNA，并反转录成cDNA，进行PCR筛选、克隆、测序，最终获得了二个小麦Mlo基因全长cDNA克隆（Ta-Mlo、Ta-Mlo1）（GenBank登录号AF384144、AF384145），利用简并引物也从易位系mRNA反转录的cDNA中扩增获得了一段与大麦Mlo蛋白高度同源的cDNA序列。对上述分离、克隆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。 2．为深入了解克隆基因是否与6VS/6AL易位系的抗病性相关，本研究将抗病易位系及感病亲本扬5经白粉菌诱导（0，12，24，48，72h）后提总RNA，转膜。进而对克隆的一系列基因进行了Northern杂交分析。结果表明，小麦病程相关蛋白1基因（Ta-Pr1）及类甜蛋白基因（Ta-TLP）为诱导表达基因（非诱导对无转录物累积），其余基因在非诱导时均有本底水平表达，但经白粉菌诱导后，表达水平明显增强，并发现各基因在经白粉菌诱导后的抗病易位系及感病亲本杨5中表达方式明显不同，证实分离、克隆的这些基因与易位系抗病性是相关的。 3．为研究克隆的基因在小麦基因组中的结构以及抗病易位系6VS上是否携有这些基因，通过提取易位系，扬5、簇毛麦、簇毛麦－硬粒小麦双二倍体DNA，进行 Southern杂交，结果表明，克隆的小麦病程相关蛋白 1 基因（TaPrl）、CyClophilin基因（Ta－Cyp）及J麦Mlo、Mlol基因（TaMlo、TaMlol）在J麦基因组中均为 2—3个拷贝，小麦类甜蛋白基因（Ta－TLP）为 l—二个拷贝，而小麦卜门．3，1．川葡聚糖苦酶（TOLMZ）和H”－ATP酶基因（TUMAH）均为单拷贝。研究发现，Ta－Prl和Ta（yp基因在扬5、易位系之间存在多态性，进一步Southern分析证实易位系6VS上携有Ta－Cyp基因。利用中国春缺体－四体系及缺失体系，己将Ta七 基因定位到小麦6A、6B、6D染色体上，将Ta－TLP定位到7B、7D染色体；并将小麦Ta－Mlo基因定位到ZAL，ZBL，ZDL的特定区域上。 4．本研究进一步通过构建表达载体，使 Ta－Mlo、TaMlo及 Ta1LP基因在大肠杆菌溶源菌 BLZI（DE／中得以表达，通过制备抗 TaMlo、TaMlo和 TaTLP表达产物的兔抗血清，对经白粉菌诱导后的抗病易位系（6VS／6AL）和感病亲本扬 5幼苗叶中表达的 Mlo、Mlo及 TLP蛋白进行兔疫印迹检测（western杂交）。结果表明，小麦叶中Ta－Mlo蛋白为膜结合蛋白；并发现仅在小麦叶经白粉菌诱导后，才能检测到该蛋白，表明Ta－Mlo蛋白为诱导表达产物。且扬5、易位系中，该蛋白在表达时问及表达量上均存在差异，暗示M。蛋白与易位系的抗病性是相关的。本研究中没有检测到 Ta－Mlo 的表达产物。研究发现，Ta－TLP蛋白存在于小麦叶细胞蛋白中，其为诱导表达产生，在诱导后的扬5、易位系中其表达量明显不同，表明该蛋白在易位系的抗病过程中有可能发挥一定作用。 5．通过田间及细胞遗传学鉴定，获得了感白粉病的小麦．簇毛麦6V缺失附加系＊n＝44）Nel石Vs2X通过 C分带及原位杂交发现感病的 6V缺失附加系中 6V染色体已丢失染色体短臂 46％的远侧端。利用现有的 12个小麦族第六群短臂 yLP探针，对该材料及抗白粉病的 6V缺失附加系uel．6Vsl）材料进行 yLP比较分析，没能找到多态性标记。需进一步寻找与之更近的分之标记，该缺失附加系材料的发现对 PmZI基因的进一步精细定位具重要作用。更多还原

【Abstract】 Powdery mildew caused by Erysiph graminis f. sp tritici, is one of the most serious disease of common wheat in China and in many countries of the world. Developing resistant varieties is the most effective way to control powdery mildew. The Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line containing powdery mildew resistance gene Pm21 developed by cytogenetic institute of Nanjing Agricultural University, which confers effective resistance to all current powdery mildew pathogens. In an attempt to understand the molecular basis of disease resistance of Pm2 1 and isolate Pm21 gene, the 6VS/6AL translocation line was used to isolate and clone powder mildew resistance related gene. 1. In this study, reverse-transcription polymerase China reaction (RT-PCR) screening approach were applied to isolate cDNA clone by using degenerate primers designed based on the AA sequence of known plant disease-resistance genes the conserved region and specific primers designed according to the known defense response gene and Mb gene of barley. Eight cDNA clones corresponding to mRNA differentially induced in resistant 6VS/6AL translocation line compared to susceptible wheat cultivar 揧angmai 5?by powdery mildew infection were isolated. Among these eDNA clones, three eDNA clones that are high homologous to barley pathogenesis - related protein lb gene, thaumatin-like protein gene and wheat 13?l .3, 1.4) glucanase gene have been isolated respectively with specific primers of known defense response gene, and by using RACE technology and screening eDNA labrory, the fall-length cDNA clones of wheat pathogenesis-related protein 1 gene (GenBank accession AF384143) and thaumatin like protein gene (GenBank accession AF384146) have been obtained; the cDNA sequences coding of wheat cycbophilin-like gene (GenBank accession AF384147) and H?ATPase like gene (GenBank accession AF384148) and the cDNA clone that has high homologous to NBS-LRR type resistance protein in rice or maize were first isolated; the two full-length eDNA clones of wheat Mb gene 3 (GenBank accession AF384144, AF384145 )have also been obtained with specific primers of barley Mb gene. The nuclectide and deduced amino acid sequences derived from all of these clones were analysied and characterized. 2. Comparison of the expression level of cloned genes in different infection times were assessed by the Northern blotting. The result showed that all cloned genes have induced expression activity, no transcription products or low level accumulation of mRNA from putative genes were observed from control (no infection), and the abundantly accumulation of corresponding mRNA transcriped from putative genes were revealed in infected translocation line and ?Yangmai 5? The obvious difference in expression time or expression level can be observed between resistant translocation lines and susceptible wheat cultivar 揧angmai 5? it implys that these cloned genes may be related with disease resistance of translocation lines. 3. To determine the genomic construction of cloned genes in wheat genome ,the translocation line and Yangmai 5 genomic DNA digested using three restriction enzyme (EcoRI, EcoRV ,HindIII) and cloned gene were labelled as probe respectively, the Southern blot indicated the wheat genome contains 2? copy of the pathogenesis- related protein 1 gene (Ta-Prl), the cyclophilin gene (Ta-Cyp) and the wheat Mb, Mlol (Ta-Mb, Ta-Mlol) gene; [? copy of the thauma更多还原