Genetic Analysis Of Wheat-Rye Derivatives Resistance To Powdery Mildew And Differential Gene Expression And Cloning

Wheat is the most widely cultivated and important food crop in the world. It is a stable food crop for about 35% of the human population, so its yield affects the supply of world food. Yield loss of wheat from disease, especially from powdery mildew, reached 10%-50%.Powdery mildew, caused by Blumeria graminis f. sp. tritici (syn. Erysiphe graminis f. sp. tritici), is one of the most damaging diseases of wheat in many areas around the world especially in China. There were many physiological races and it was inevitable to loss the resistance to disease. Utilization of resistant cultivars and explore of mechanism of resistance disease were the most effective, economical and environmentally safe approaches to control plant diseases. In this paper, genetic analysis and construction of SSH (Suppression Subtractive Hybridization) were applied to study wheat-rye derivatives. The results are as followed.1 Wheat-rye derivatives N9436-1 and N9436-2 were analyzed using investigation of agronomic characters, identification of resistance to powdery mildew, cytology, genomic in situ hybridization (GISH), biochemistry and molecular markers. It was showed that N9436-1 and N9436-2 were wheat-rye 1R and 6R additional lines respectively. And they were immune or high resistant to powdery mildew. N9436-1 had multi-spikelet and N9436-2 had purple stem.2 cDNA libraries of wheat germplasm N9436-1 resistant to powdery mildew were constructed using SSH. About 550 clones were picked randomly from the forward and the inverse libraries each.3 A total of 140 positive clones were randomly chosen from the forward SSH-cDNA library and sequenced. After screening repeat and redundant sequences, 94 ESTs were acquired and were submitted to GenBank. Accession numbers of GenBank were from EX567357 to EX567450 and dbEST-Id from 50073321 to 50073414. Nucleic acid and protein homology search were performed using the BLAST (Basic Local Alignment Search Tool) program with the default settings at NCBI website (http://www.ncbi.nlm.nih.gov). BlastX results in nr-protein database revealed that 49 ESTs were highly homologous with known proteins involved in primary metabolism (2%), energy metabolism (24%), cell structure (2%), transcription (2%), protein synthesis and processing (16%), transport (4%), signal transduction (4%) and disease resistance and defense (30%). And ESTs unknown function occupied 16%. There were 22 ESTs associated with powdery mildew resistance, including 6 ESTs for signal transduction, 2 for hypersensitive necrosis reaction (HR) system, and 14 for systemic acquired resistance (SAR) system, respectively.4 A total of 120 positive clones were randomly chosen from the inverse SSH-cDNA library and sequenced. After screening repeat and redundant sequences, 59 ESTs were acquired and were submitted to GenBank. Accession numbers of GenBank were from EX567298 to EX567356 and dbEST-Id from 50073262 to 50073320. Nucleic acid and protein homology search were performed using the BLAST (Basic Local Alignment Search Tool) program with the default settings at NCBI website (http://www.ncbi.nlm.nih.gov). BlastX results in nr-protein database revealed that 23 ESTs were highly homologous with known proteins involved in primary metabolism (9%), energy metabolism (44%), transcription (4%), transport (9%), signal transduction (4%) and disease resistance and defenses (26%). And ESTs unknown function occupied 4%. There were 7 ESTs associated with powdery mildew resistance, including 1 for signal transduction and 6 for SAR system, respectively.5 Comparing the EST sequences among the SSH-cDNA libraries, gene expression patterns of 8 ESTs in resistance reaction of powdery mildew were studied by using semi-quantitative RT-PCR (reverse transcription PCR). They were pathogenisis-related protein(Z25-1), glutathione-S-transferase(Z440-1), peroxidase(Z208), putative cytochrome P450(Z219-1), stress responsive protein(F76), phosphoribulokinase(F105), glyoxalase(F110-1), hydroxypyruvate reductas(eF167). It was showed that they belonged to inducible expression genes and were involved in the resistance to powdery mildew. Pathogenisis-related protein, glutathione-S-transferase, peroxidase and phosphoribulokinase were up-regulated genes, while cytochrome P450 and glyoxalase were down-regulated genes.6 By in silico cloning, 5 ESTs were extended. And according to the extended sequences, primers were designed to verify the results by RT-PCR. It was showed that glutathione-S-transferase was 875bp long with a complete open reading frame encoded a 229 amino acid protein. Then through analysis of blastp, glutathione-S-transferase had N-terminal and C-terminal domains. It was showed that this sequence also contained thioredoxin fold and superfamily GST domain by InterProScan tool. From the above, a gene fragment of GST was cloned. It was located on cell nuclear, its isoelectric point and molecular weight were 4.97 and 71910.41Da respectively.