| Wheat powdery mildew caused by Blumeria graminis f. sp. tritici is a devastating disease in wheat growing countries worldwide. Resistance cultivars offer the most effective and safe approach to control the disease. However, the use of single or few Pm genes eventually leads to a rapid development of correponding virulence genes in the pathogen, resulting in breakdown of cultivars’resistance. Thus, it is foundational to identify novel Pm genes and dissect resistance mechanism for the control of wheat powdery mildew.Wheat landrace may contain rich resistance genes, which is very valuable to explore resistant cultivars and identify new resistance genes against powdery mildew. Hongyoumai, one wheat landrace, bears good resistance to powdery mildew. Genetic analysis revealed that it carried one dominant Pm gene. In the present thesis, SSR markers were employed to locate Pmhym gene. Meanwhile, Affymetrix genechip analyses were employed for expression profiling in response to Blumeria graminis f. sp. tritici infection in homologous resistant and susceptible F3 progeny derived from Hongyoumai and Yumai13, respectively, along with verification of differentially expressed genes using real time quantitative RT-PCR (qRT-PCR) analyses. Based on the genechip results, one up-regulated target gene in resistance progeny was further in silico cloned and characterized. The main contents and results were as follows:1. Chromosome location of Pmhym geneBulked segregate analysis (BSA) was used to establish the resistant and susceptible gene pools from F3 homologous progeny of Hongyoumai×Yumai13.250 pairs of primers were employed to screen the two parents and two pools. Primers which can amplify the same polymorphic bands between parents and pools will be further selected to detect an F2 population. Mapmaker/EXP (VERTION3.0b) analysis was then applied, and the results revealed three markers identified to be linked to Pmhym, the order of which was:Xwmc526-31.6cM - Xgwm577 - 25.6cM - Pmhym - 14.3cM - Xwmc232. Three markers were found to be located on the long arm of 7B chromosome, which indicated that gene Pmhym might be also located on chromosome 7BL.2. Identification of differential expressed genes induced by Blumeria graminis using genechip. Four F3 homologous resistant progeny of Hongyoumai×Yuma13 were selected as the resistance pool, with four susceptible progeny as the susceptible pool. Two treatments were set for both of the two pools:uninoculated,24 h after inoculation by Blumeria graminis. Data obtained from hybridization of cRNAs of the samples crossed with Affymetrix wheat genechip were normalized, and three groups were categorized including pre- and post-induction of the resistant, pre- and post-induction of the susceptible, and post-induction of the resistant and susceptible, respectively. Base on the differentially expressed genes,22 primer sets were designed and used for qRT-PCR validation. The qRT-PCR confirmed the reliability and reproducibility of the data revealed by genechip.The gene measure up to the following criterions:P (present) hybridization sigal in tester, change p-value<0.001 and ratio≥2 or≤0.5, it will be considered as differentially expression. 2728 differential genes were identified between uninoculated and infected 24 h in resistant plants,39.71 percent genes are disease/defense ones in 408 genes with known function.2631 differential genes were identified between uninoculated and infected 24 h in susceptible plants,37.33 percent genes are disease/defense ones in 442 genes with known function. It indicated that metabolisms related to resistance were induced by Blumeria graminis.Comparison of resistance- and susceptibility-related expression profiling in resistant and susceptible wheat infected by Bgt, indicated that three categories of induced transcripts were obtained including resistance-specific, susceptibility-specific and basal defense transcripts. It was found that SA (salicylic acid), JA (jasmonate) and ET (ethylene) signaling pathways related genes were differentially expressed, thus implied that the three signaling pathways might be involved in the wheat defense response against Bgt infection. In the susceptible plants, genes involved in ABA (Abscisic acid) signaling pathway might be inhibitive to the abovementioned three pathways, thus resulting in susceptible reaction. The number of genes involved in ROS (Reactive oxygen species) metabolism was more in the resistant plants, indicating possible involvement in resistance reaction. And genes involved in disease/defense, signal transduction were generally up-regulated, suggesting their implication in defense response.3. Cloning of a wheat germin-like protein geneOne probes Ta.169.1.S1_x_at up-regulated in the inoculated resistant plants was selected for in silicon extension. Primers were then designed based on in silico cloned sequence to amplify cDNA of the corresponding gene. One germin-like protein encoded gene named TaGLP5 (855 bp) with a full open reading frame was obtained, and deposited in GenBank with accession of FJ594470. BLASTX analysis demonstrated that the encoded TaGLP5 protein belonged to a Cupin2 superfamily, with 15 homologous sequences (50-63%). The analyses of multiple alignment and phylogenetic tree indicated that TaGLP5 belongs to different branches from known germin-like protein. TaGLP5 was located on chromosome 5A with a PCR-based method using a set of Nulli-tetrasomic lines of "Chinese Spring". qRT-PCR analysis showed that TaGLP5 transcript was induced in response to Bgt infection, with higher expression in resistant plants than in the susceptible pre-24 h inoculation. It was speculated that TaGLP5 might be involved in wheat defense response against Bgt. |
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