Construction Of H.villosa CDNA Library, Cloning And Function Analysis Of Wheat (Triticum Aestivum) Resistance Relative Gene

Powdery mildew, caused by Erysiphe graminis DC. f.sp.tritici Marchal, is one of the major diseases of common wheat worldwide. Development and utilization of wheat cultivars with resistance gene is an efficient, economic and environmentally safe strategy to control powdery mildew. Many powdery mildew resistance genes had already lost their resistance effect because of variation of prevalent races. Pm21, originally introduced into wheat from H. villosa through chromosome translocation of 6VS/6AL, is an effective powdery mildew resistance gene with broad spectrum in the world and has a good promise in wheat breeding. So cloning of Pm21 gene and powdery mildew resistance-related genes will play an important role in future disease resistance breeding.Construction of cDNA library is a prerequisite for gene cloning. But it is difficult to construct and screen a cDNA library from common wheat because of its large genome. In this study, a cDNA library was constructed using cDNA from leaves of diploid Haynaldia villosa (2n=14, VV) inoculated with Erysiphe graminis. The cloning vector was an expression plasmid vector pSPORT1 which was introduced into E.coli DH10B. The cDNA library contains about 30,0000 recombinant cDNA clones, with average insert size of 1.6kb and is successfully screened with the probes of wheat glutathione reductase gene (Ta-GR), a blue copper-binding protein (BCB) gene, a glutathione-S-transferase (GST) gene and other genes cloned by ourselves.RT-PCR screening approach using degenerate primers designed based on conserved domains of plant resistance disease genes and specific primers designed according to the ESTs which had been mapped onto wheat homologous group 6 was applied to isolate resistant related gene. Ta-RPM and Ta-Mla isolated from translocation line with specific primers are high homologous to barley NBS-LRR resistant protein b7 (AAB96982.1) and barley CC-NBS-LRR resistance protein MLA13 (AF523682.1) , respectively. TH8 and TH12 obtained by degenerate primers have high homology to Arabidopsis Ser/thr protein kinase and human MDHC gene, respectively. Southern blot with Chinese Spring nullisomic/tetrasomic lines was used to assign the genes of TH8 and TH12 to chromosome 5D and 3D, respectively. The nucleotide and deduced amino acid sequence of the four clones were analyzed and characterized.