【作者】 李岩；

【导师】 王洪刚； 封德顺；

【作者基本信息】 山东农业大学 ， 作物遗传育种学， 2007， 硕士

【摘要】 小麦是世界上最重要的粮食作物之一。小麦白粉病是由白粉菌Blumeria graminis f. sp. tritici .(Bgt)引起的世界性真菌病害。利用小麦自身的抗性是控制白粉病最有效、经济和安全的途径。克隆植物抗病基因,通过转基因的方法增强植物抗性,是控制植物病害的新方法。为了解小麦抗白粉病的机制,本文开展了对小麦白粉病抗性基因类似物的克隆研究。Pm3b是世界上第一个被克隆的小麦白粉病抗性基因,在采用图位克隆法克隆Pm3b的过程中,发现Pm3b是成簇排列的NBS-LRR类抗病基因家族的一员。克隆Pm3b所在基因家族的其它成员,对于研究NBS-LRR类抗病基因的进化及抗病机制具有重要的意义。本研究以近等基因系Ulka/XX194/8*Chancellor(Pm2)和Chancellor为材料,利用Pm3b与其它已克隆抗病基因的保守序列设计简并引物,利用PCR为基础的同源克隆法获得11个抗病基因类似物(RGA),并利用生物信息学软件进行了分析,同时对长片段扩增的体系进行了优化。获得的主要结果如下:1.与普通PCR相比,长片段PCR需要较高的pH值,TaKaRa公司生产的LA Taq酶所带GC buffer的pH值较低,需适当调高其pH值才可以从复杂基因组DNA中高效扩增长片段。2.培养温度对于长片段克隆的稳定传代有较大影响,长片段克隆在37℃条件下培养容易引起插入片段的全部或部分缺失,30℃条件下培养则有助于长片段克隆的稳定传代。3.共获得11个RGA,且均已在GenBank注册,登录号为EF157980—EF157990。所获得的11个RGA与Pm3位点的复等位基因有很高的相似性,其中3个是假基因,8个是蛋白质编码基因。种系分析发现,8个蛋白质编码基因组成一个单系群,它们与Pm3位点的复等位基因是并系关系。由于PCR扩增所用引物主要参考Pm3b设计,所以推测11个RGA来自于Pm3所属基因家族。4.大多数正向选择位点分布在LRR区,与其它结构域相比,LRR区承受着更大的正向选择压力。5.初步推测NBS-LRR类抗病基因的进化模式如下:当抗病基因的祖基因经复制而出现多个拷贝以后,大多数进化事件首先发生在LRR区,随后,发生在其它区的进化事件所占比例会逐渐提高;发生在LRR区的进化事件首先发生在β-折叠以外的其它结构上,其次才发生在β-折叠上。更多还原

【Abstract】 Wheat (Triticum aestivum L., 2n=42, AABBDD) is one of the most important food crops in the world. Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a prevalent world-wide wheat disease. Cultivation of varieties with natural resistance is the most effective, economic and safe way to control powdery mildew disease. It is a new strategy for disease controlling to clone the resistance genes and transfer it into plants for the improvement of disease resistance.Pm3b is the first powdery mildew resistance gene isolated from hexaploid wheat. Pm3b is a member of a large NBS-LRR class gene family cluster. The isolation of other members of the gene family plays a very important role in the study of the evolution of NBS-LRR class resistance gene and the mechanism of disease resistance. In this study, wheat near-isogenic lines in Chancellor background Ulka/XX194/8*Chancellor (Pm2) and Chancellor were employed and degenerate primers were designed based on the conservation of 5’-terminus and 3’-terminus of Pm3b. Eleven resiatance gene analogues (RGAs) were acquired by PCR-based cloning in this study, and the characteristic of them were analyzed by bioinformatics programs. And meanwhile, we optimized the PCR conditions for long products. The main results were as follows:1. In comparison with the amplification of short fragment, the pH value of long PCR condition should be higher. But the pH value of GC buffer bought with LA Taq from TaKaRa was not high enough and should be adjusted higher properly so as to amplify long DNA fragment effectively from high-complexity templates.2. Temperature played an important role in the stable multiplication of the bacteria clone with long fragments. The bacteria clone with long fragment insertion was prone to lose its insertion completely or partially when multiplying in 37℃, but under the condition of 30℃, the clone would be more stable.3. Eleven RGAs were acquired in this study, and registered in GenBank respectively (accession number: EF157980-EF157990). The 11 RGAs shared high similarity with Pm3 alleles. And among them, three RGAs were pseudogenes and the others were protein encoding genes. Phylogenetic analysis revealed that the eight protein encoding genes formed a monophyletic group, and they were paraphyletic with Pm3 alleles. Given that the primers were designed mostly from Pm3b, we inferred that the 11 RGAs were from the large Pm3 family.4. Most positively selected sites distributed in LRR domain, which suggested that LRR domain bore more positive selection pressure than other domains.5. We preliminarily inferred that the evolution mode of NBS-LRR resistance gene was as follows: after the ancestor gene diverged, the majority of evolution events occurred in LRR domain at first, and then, after a period of time, the proportion of evolution events occurred in other domains would increase; the evolution events in LRR domain occurred in other secondary structures firstly, and then inβ-sheet.更多还原