The Study Of Cloning Wheat Powdery Mildew Resistance Related Gene

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.