【作者】 张丽娜；

【导师】 于玲； 牛吉山；

【作者基本信息】 西北师范大学 ， 植物学， 2005， 硕士

【摘要】 小麦白粉病是严重危害小麦生产的病害之一,它是由白粉菌(Blumeria graminis(DC.)E. O. Speer f. sp. tritici Em. Marchal, Bgt)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。小麦-黑麦1BL/1RS易位系99/2439对白粉病表现为近免疫,对其抗白粉病相关基因进行克隆研究不仅有利于阐明其抗病机制,而且对抗病育种有重大意义。本研究进行了以下几方面的研究: 1.构建cDNA文库是克隆基因的基础,本研究用质粒载体pGEM-T构建了经白粉菌诱导1、4、12、24、48和72 h的小麦-黑麦1BL/1RS易位系99/2439叶片cDNA文库一个。文库寄主菌为E. coli DH10B,诱导文库平均插入片段为1kb左右。主要分布在800 bp-2 Kb之间。 2.用RACE技术对经白粉菌诱导24 h的小麦-黑麦1BL/1RS易位系99/2439叶片的cDNA进行扩增,获得了小麦类受体蛋白激酶基因(RLK)全长cDNA克隆(TaLRK)(GenBank登录号:AY584533)。蛋白质一级结构分析表明,该基因与小麦蛋白激酶LRK19和小麦抗锈病激酶LRK10高度同源。以小麦肌动蛋白基因(Actin)作为对照,利用半定量反转录PCR(Semi-QRT-PCR)技术,研究了TaLRK基因的表达,发现该基因在经白粉菌诱导后的叶片中表达明显增强。在非诱导的茎中有痕量的表达产物,在穗、根中不表达,不同时期的叶片中表达明显比茎中强。 3.用RACE技术对经白粉菌诱导24 h的小麦-黑麦1BL/1RS易位系99/2439叶片的cDNA进行扩增,获得了与小麦和大麦Mlo同源性很高的基因序列(TaMlo-Bld)(GenBank登录号:AY584534)。利用半定量RT-PCR技术,研究了TaMlo-Bld基因的表达,发现该基因在白粉菌诱导后的叶片中表达稍微有所增强。在叶、茎、根中均表达,在穗中不表达。诱导的根中表达明显增强。 4.用RACE技术对经白粉菌诱导24 h的小麦-黑麦1BL/1RS易位系99/2439叶片的cDNA进行扩增,获得了与大麦HvEDR1基因高度同源的小麦TaEDR1基因全长cDNA克隆(GenBank登录号:AY743662)。首次提供了证明普通小麦中存在EDR1基因同源物的分子证据。利用半定量RT-PCR技术,研究了TaEDR1基因的表达,发现该基因在白粉菌诱导后的叶片中表达明显增强。在叶、穗、茎、根中均有表达。更多还原

【Abstract】 Powdery mildew caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici, is one of the most severe diseases of common wheat in china and in many countries of the world. Developing resistant varieties is the most effective way to control powdey mildew. A wheat-rye lBL/lRS alien translocation line 22/2439 is resistant to wheat powdery mildew in China. Cloning the powdery mildew resistance related genes is useful for understanding the resistance mechanism and disease resistance breeding.1. To construct cDNA library is a prerequisite for gene cloning. In this study, a cDNA library was constructed using leaf mRNA of wheat-rye 1BL/1RS translocation line 22/2439 that was induced by Bgt. The cloning vector was a plasmid pGEMT-easy vector, which was introduced into E. coli DH10B. The average insert size is 1.2 Kb, ranging basically from 800 bp to 2.0 Kb.2. RACE technique was successfully used to amplify of a cDNA from leaves of wheat-rye 1BL/1RS alien translocation line 22/2439 induced with Bgt. A full-length cDNA (GenBank accessionAY584533) clone was obtained, it belongs to a receptor-like kinase gene family in wheat, and named as TaLRK. Protein database search revealed that it is high homologous to wheat LRK19 protein kinase, and wheat rust resistance kinase LRK10. Expression analysis of TaLRK gene was carried out by Semi-QRT-PCR, using wheat Actin gene as a control. The expression analysis of TaLRK gene in leaves showed that the transcribing was enhanced significantly by the pathogen infection. The transcription levels of TaLRK gene in leaves were relatively higher than in stem, there was very little amount transcripts in stem. no expression in spike and root.3. RACE technique was successfully used to amplify of a cDNA from leaves of wheat-rye lBL/lRS alien translocation line 22/2439 induced with Bgt.A clone obtained is high homologous to wheat Mlo and barley Mlo proteins, It was named 7aMlo-B1d(GenBank accession AY584534). Expression analysis in different organs in wheat of TaMlo-Bld gene was carried out by Semi-QRT-PCR . The expression analysis of TaMlo-Bld gene in leaves showed that the transcribing was enhanced slightly by the pathogen infection. TaMlo-Bld gene was expressed in leaf, stem, root, no expression in spike.4. RACE technique was successfully used to amplify of a cDNA from leaves of wheat-rye 1BL/1RS alien translocation line 22/2439 induced with Bgt. A full-length cDNA clone obtained is high homologous to barley EDR1 gene, It was named as TaEDR1 (GenBank accession AY743662). This firstly provided a molecular evidence to demonstrate that EDRl homolog exists in common wheat. Expression analysis of wheat TaEDR1 gene in different organs was carried out by Semi-QRT-PCR. The expression analysis of TaEDR1 gene in leaves showed that the transcribing was enhanced significantly by the pathogen infection. TaEDR1 gene was expressed in leaf, spike, stem, and root.更多还原