Cloning, Characterization, Mapping And Expression Analysis Of Powdery Mildew Resistance Relative Genes In Wheat

Powdery mildew caused by Erysiph graminis f. sp tritici, is one of the most serious disease of common wheat in China and in many countries of the world. Developing resistant varieties is the most effective way to control powdery mildew. The Triticum aestivum-Haynaldia villosa 6VS/6AL translocation line containing powdery mildew resistance gene Pm21 developed by cytogenetic institute of Nanjing Agricultural University, which confers effective resistance to all current powdery mildew pathogens. In an attempt to understand the molecular basis of disease resistance of Pm2 1 and isolate Pm21 gene, the 6VS/6AL translocation line was used to isolate and clone powder mildew resistance related gene. 1. In this study, reverse-transcription polymerase China reaction (RT-PCR) screening approach were applied to isolate cDNA clone by using degenerate primers designed based on the AA sequence of known plant disease-resistance genes the conserved region and specific primers designed according to the known defense response gene and Mb gene of barley. Eight cDNA clones corresponding to mRNA differentially induced in resistant 6VS/6AL translocation line compared to susceptible wheat cultivar 揧angmai 5?by powdery mildew infection were isolated. Among these eDNA clones, three eDNA clones that are high homologous to barley pathogenesis - related protein lb gene, thaumatin-like protein gene and wheat 13?l .3, 1.4) glucanase gene have been isolated respectively with specific primers of known defense response gene, and by using RACE technology and screening eDNA labrory, the fall-length cDNA clones of wheat pathogenesis-related protein 1 gene (GenBank accession AF384143) and thaumatin like protein gene (GenBank accession AF384146) have been obtained; the cDNA sequences coding of wheat cycbophilin-like gene (GenBank accession AF384147) and H?ATPase like gene (GenBank accession AF384148) and the cDNA clone that has high homologous to NBS-LRR type resistance protein in rice or maize were first isolated; the two full-length eDNA clones of wheat Mb gene 3 (GenBank accession AF384144, AF384145 )have also been obtained with specific primers of barley Mb gene. The nuclectide and deduced amino acid sequences derived from all of these clones were analysied and characterized. 2. Comparison of the expression level of cloned genes in different infection times were assessed by the Northern blotting. The result showed that all cloned genes have induced expression activity, no transcription products or low level accumulation of mRNA from putative genes were observed from control (no infection), and the abundantly accumulation of corresponding mRNA transcriped from putative genes were revealed in infected translocation line and ?Yangmai 5? The obvious difference in expression time or expression level can be observed between resistant translocation lines and susceptible wheat cultivar 揧angmai 5? it implys that these cloned genes may be related with disease resistance of translocation lines. 3. To determine the genomic construction of cloned genes in wheat genome ,the translocation line and Yangmai 5 genomic DNA digested using three restriction enzyme (EcoRI, EcoRV ,HindIII) and cloned gene were labelled as probe respectively, the Southern blot indicated the wheat genome contains 2? copy of the pathogenesis- related protein 1 gene (Ta-Prl), the cyclophilin gene (Ta-Cyp) and the wheat Mb, Mlol (Ta-Mb, Ta-Mlol) gene; [? copy of the thauma...