【作者】 丁国华；

【导师】 秦智伟；

【作者基本信息】 东北农业大学 ， 蔬菜学， 2004， 博士

【摘要】 由古巴假霜霉菌（Pseudoperonospora cubensis Rostow）引起的黄瓜霜霉病（Downy mildew）是当前严重影响黄瓜生产的主要病害之一，1868年首次被发现，近年来日趋严重。防治霜霉病可采取喷施杀菌剂和高温闷棚等措施，但根本途径是培育抗霜霉病的黄瓜优良品种。克隆和标记黄瓜霜霉病抗病基因，则是当前利用分子技术进行抗病育种工作的基础。本试验采用基于抗病基因同源序列（Resistance gene analog, RGA）克隆和标记基因的策略，主要进行了以下3方面的研究：（1）依据抗病基因的NBS保守域设计简并引物，从黄瓜基因组DNA中克隆RGA；（2）构建黄瓜抗感霜霉病分离群体，并用于从上述克隆到的RGA中筛选黄瓜霜霉病抗病基因的连锁标记；（3）利用获得的黄瓜RGA分析黄瓜的种质资源，考察RGA在黄瓜抗病种质资源上的聚类分析效果。本试验首先参照文献报道，引用和设计了12条对应NBS类保守域P-loop、kinase2、kinase3a和HD等4个亚结构域的简并引物，采用SDS方法提取了黄瓜栽培品种津春4号和649的基因组DNA作为模板，通过PCR方法以及琼脂糖凝胶回收纯化技术，分别获得约270bp、340bp和540bp的目的扩增产物，将其与pMD18-T载体连接并转化JM109大肠杆菌，经过蓝白斑初步筛选，各自随机挑取72个阳性重组克隆进行菌培养和质粒提取，经过限制性酶切和PAGE方法分类，测序了其中60个克隆，经过在GenBank上使用Blast搜索和比对同源序列，最终确定其中15个克隆为黄瓜RGA片段，分别命名为CsRGA1～15，提交GenBank，获得登录号分别为AY555482~AY555495和AY545993。其中CsRGA5包含全部上述4个亚结构域，长度520bp；CsRGA2包含3个亚结构域，长度336bp；其余CsRGA只包含2个亚结构域，长度168~288bp。10个CsRGA为可完全翻译序列，5个CsRGA内部含终止密码，为不可完全翻译序列。使用PCR方法验证了全部CsRGA在供试样本基因组的存在。10个可通读CsRGA与公布的甜瓜RGA之间存在较高的同源性，显示出两者同属的亲缘关系。为从CsRGA中筛选黄瓜霜霉病抗病基因标记，分别以631和L18为感病亲本、649和129为抗病亲本，构建了4个以631×649和L18×129为杂交组合的霜霉病抗感性状的自交和回交分离群体，使用基于CsRGA1~15设计的特异引物，在上述群体中进行常规PCR扩增，在琼脂糖凝胶上获得多态性电泳谱带；为考察筛选引物的标记效果，同时进行了已公布黄瓜霜霉病抗病基因SCAR标记SCBC519和SCBC526的PCR扩增和分析。群体内各单株的DNA采用SDS微量简易提取法提取，抗病等级采用子叶点滴人工接菌法鉴定。连锁分析使用MAPMAKER/EXP3.0版本分析软件。结果显示：L18×129 F2回交分离群体（L18为回交亲本）、631×649 F2自交分离群体（单株数86）和L18×129 F2自交分离群体（单株数77）的霜霉病抗感性状都有合适的分离比值（1:1和1:3），在L18×129 F2自交<WP=10>分离群体上得到较好的连锁标记，从15个CsRGA中筛选到与抗病基因dm连锁不十分密切的CsRGA3标记。在dm和CsRGA3之间还检测到黄瓜白粉病抗病基因pm的存在，显示了dm和pm存在连锁关系。本试验为研究植物RGA在种质资源分析方面的效果，特别是标示抗感病品种资源的作用能力，选择了24个黄瓜主要栽培品种进行标记聚类分析。所使用的标记为CsRGA3、BC519和BC526等3种标记（后两者为已公布的dm的RAPD标记），采用普通PCR方法扩增产物，琼脂糖凝胶电泳观察多态性谱带，并使用MVSP 3.13分析软件对谱带进行UPGMA聚类分析，以70％相似性（similarity）为域值，将供试品种划分为4类。与黄瓜霜霉病人工接菌鉴定结果相对照发现，3个标记可以明确标示出高感霜霉病黄瓜品种（长春密刺和L18），但对中抗和高抗类型分辨不够清晰。表明此种专项种质资源的分析方法基本可行。本试验在实验方法上有所创新。首次采用PCR-PAGE方法对RGA的克隆进行分类，与四碱基酶切分类方法相比较，该方法能够全面分析待测的克隆核苷酸序列，PCR扩增不受载体序列干扰、无需酶切鉴定可以直接去除非重组克隆，同时还可以对重组克隆进行是否为目的片段克隆的PCR验证。该方法在对电泳谱带的分析上也比较简便，只需对目的电泳带进行长度的归类，借助图象扫描分析软件可轻易完成。综上所述，本试验证实了NBS类型RGA在黄瓜基因组中广泛存在，并与甜瓜RGA有较高同源性；本试验还初步验证了黄瓜RGA同某些抗病基因之间可能存在的连锁关系；本试验还首次尝试使用RGA的特异引物分析植物品种间的遗传关系，初步证明黄瓜RGA对抗病种质有一定的标示作用。更多还原

【Abstract】 Cucumber downy mildew, which is caused by Pseudoperonospora cubensis Rostow, is one of main diseases in cucumber product. It was firstly discovered in 1868, and had a serious development recently. Methods of control downy mildew included utilizing medicine and closing a greenhouse at high temperature, but were ultimately to produce resistant varieties. So cloning and marking the resistance gene of the downy mildew in cucumber are fundaments for disease resistance breeding of cucumber.The study consisted of three parts: (1) the resistance gene analogs of cucumber were isolated by performing PCR, in which degenerate primers designed according to conservative domain of resistance genes were used; (2) the separate population with resistant and susceptible traits for downy mildew were constructed and used for screening markers on downy mildew resistance gene among the cloned RGAs; (3) the novel RGAs isolated from cucumber were employed for investigation of cucumber germplasm resource and their potential of cluster analysis was tested.Firstly, the genome DNA as template from varieties JINCHUN 4 and 649 were extracted by using SDS method, and twelve degenerate primers were designed based on four substructure motifs, P-loop, kinase2, kinase3a and HD, within NBS conserved domain referring to some reports, and amplifying products, which spanned approximately 270bp, 340bp and 540bp, were obtained by performing PCR and purification technique, which were cloned into pMD18-T vector and transformed E. coli JM109. After blue and white plaque preliminary screening, each seventy-two recombinant clones were randomly picked and their plasmids were prepared and were classed by restriction endonuclease digesting and PAGE method. Sixty clones were sequenced and their homologous sequences were searched on GenBank with Blast. Finally, fifteen clones were distinguished as RGA of cucumber and named CsRGA1~15, which were submitted to GenBank and provided accession numbers as AY555482~AY555495 and AY545993. Among the CsRGAs, the CsRGA5 contains all of four substructure motifs and spans 520bp, and CsRGA2 <WP=12>involves three ones of them and spans 336bp, and other CsRGAs only include two ones of them and span 168~288bp extent. Ten CsRGAs have open reading frames (ORFs) and other five ones don’t have because of stop codons in them. Performing PCR supported the truth of all CsRGAs exist in the genome of tested cucumbers. The ten CsRGAs having ORFs showed high similarities with the announced RGAs of Cucumis melo, which displayed a close relationship as both species are in the same genus.For screening markers of downy mildew resistance gene from the CsRGAs, four separate populations with resistant and susceptible traits for downy mildew were constructed. Two separate populations were F2 intercross and two other populations were F2 backcross. Many polymorphic bands were obtained on agarose gel electrophoresis pattern using special primers designed based on CsRGA1~15 sequences and performing PCR in above populations. Meantime, the PCR of SCBC519 and SCBC526, both of which were the markers for dm, were performed. The genome DNA of the individuals were extracted by SDS-mini-extracting method, and the level of disease resistance was evaluated by inoculating into the cotyledons of the individuals, and the linkage analysis was carried out by MAPMARKER/EXP version 3.0 program. The results showed that the separate ratios of both disease resistant and susceptible traits in the populations, which were F2 backcross one of L18×129 with L18 as backcross parent, F2 intercross one of 631×649 (the individual amounted to eighty-six), and F2 intercross one of L18×129 (the individual amounted to seventy-seven), matched up to Mendel’s law; the linkage marker, CsRGA3, to the dm was picked out from fifteen CsRGAs and was not very close to the dm; the pm locus that being powdery mildew resistance gene existed between CsRGA3 and dm, which was detected and showed the linked relationship of both pm and dm.For the sake of knowing the research potential of analyzi更多还原