【作者】 高倩；

【导师】 刘大群； 王海燕；

【作者基本信息】 河北农业大学 ， 植物病理学， 2008， 硕士

【摘要】 由小麦叶锈菌(Puccinia triticina)引起的小麦叶锈病是影响世界小麦稳产高产的重要病害之一,也是影响我国小麦生产的重要因素。持续筛选抗病基因和培育抗病品种是防治小麦叶锈病发生的主要手段。小麦抗叶锈病基因Lr35最初来源于拟斯卑尔脱山羊草(Aegilops speltoides),通过与Triticina.monococcum的二倍体回交将其转移到小麦品种马奎斯(Marquis)中。该基因位于2B染色体上,与Sr39紧密连锁。其抗性在二叶期开始表达,六叶期完全表达,是一个应用潜力很大的由主效基因控制的抗病基因。抗病基因类似序列(Reistance gene analogys,RGAs)技术是利用抗病基因具有保守结构域的特性,通过人工合成引物对基因组DNA进行定点扩增,在全基因组水平上检测DNA变异。本试验以含小麦抗叶锈病基因Lr35的近等基因系材料TcLr35和感病亲本Thatcher以及其它小麦抗叶锈病近等基因系材料进行RGA分析。主要结果如下:根据已克隆的植物抗病基因普遍具有的NBS和LRR结构域设计49条RGA引物,组成260对引物组合,从近等基因系TcLr35中扩增获得4条特异性片段。其中引物对Pto-kin1IN/XLRR-INV1从近等基因系TcLr35中扩增获得一条747bp的稳定特异性片段。以本实验室保存的49个小麦抗叶锈病近等基因系材料进行扩增,发现此条带仅在TcLr35中存在,而在其他48个小麦抗叶锈病近等基因系材料及感病亲本Thatcher中均未出现,说明该片段特异性很强。对该特异性片段进行序列测定与分析,经BLAST比较,表明该片段与许多已克隆出来的小麦基因序列相似:与Triticummonococcum DV92的BAC克隆231A16有84%的同源性,与Triticum urartu clone BAC210J24有96%同源性;与Triticum turgidum subsp.durum clone BAC 221H19有90%同源性;与Triticum urartu clone BAC 41C8有89%同源性;与Triticum urartu clone BAC292N12有88%同源性等。对该特异性片段进行蛋白质序列比对,未发现与其于已知功能小麦片段有较高同源性。更多还原

【Abstract】 Wheat leaf rust caused by Puccinia triticina is one of the most important diseases affected the wheat production world widely. Utilization of genetic resistance is the most economical and environment friendly strategy for disease control. Wheat leaf rust resistance gene Lr35 mapped on chromosome 2B was derived from Aegilops speltoides and was transferred to the wheat cultivar Marquis through baekcrossing with diploid Triticina monococcum. The gene closely links with the wheat stem rust resistance gene Sr39. Its resistance to the disease first appears at the growth stage with two leaves, and completely expresses with six leaves. It is a very effective leaf rust resistance gene which has great potential to be used on wheat productionReistance gene analogys (RGAs) is a technique which can detect the DNA mutation from the genome scale by utilizing the character of the resistance gene conserved region and amplify the genome DNA specifically through man-made synthesing primers. In this experiment, near-isogenic line (NIL) TcLr35 carrying Lr35, its susceptible parent Thatcher and the other NILs were served as material to perform RGA analysis. The main results were as follows:Using the 260 primer pairs from 49 individual RGA primers which based on the conserved domains (NBS、LRR etc) of the known resistance genes, 4 specific fragments were found in TcLr35. A steady and specific747bp fragment was found in TcLr35 by primer pair Pto-kin1IN/XLRR-INV1. The primer pair then was used to amplify resistant homologous sequences in the 49 near isogenic lines (NILs) of wheat leaf rust resistance. The specific fragment was only found in TcLr35, but never in the other 48 near isogenic lines and Thatcher. The result indicates that the specific fragment is high differential. The specific fragment was isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. It has the high homology with other wheat gene sequences cloned: 84% identical with Triticum monococcum DV92 clone BAC 231 A16, 96% identical with Triticum urartu clone BAC 210J24, 90% identical with Triticum turgidum subsp. durum clone BAC 221H19, 90% identical with Triticum urartu clone BAC 41C8, 88% identical with Triticum urartu clone BAC 292N12 etc. The BLASTx analysis indicates that there are no wheat DNA fragments showing high homologous with the specific fragment.更多还原