| Wheat powdery mildew disease, caused by Blumeria graminis f.sp. tritici, is one of the most important wheat diseases affecting the production of wheat all over the world. Compared with the chemical control methods, cultivation of wheat cultivars with resistance is the most effective method for the powdery mildew control. Cloning of disease-resistance related genes will not only provide gene resources for wheat by improvement genetic engineering, but also help us understand the molecular aspects of the Pm21 gene mediated broad spectrum resistance to powdery mildew disease.In the previous research, GeneChip microarray analysis and cDNA library screening was used to clone genes related to the Pm21-mediated powdery mildew resitance in Haynaldia villosa L.. A full length cDNA sequence of the Hv-CMPG gene was cloned (Liu, 2007). The gene was physically mapped to chromosome 6V short arm of Haynaldia villosa, where the Pm21 was located. In vitro ubiquitination activity assay proved that Hv-CMPG had the E3 ligase activity. Semi RT-PCR analysis and transient expression assay indicated Hv-CMPG contributed to the powdery mildew resistance. Hv-CMPG was transformed into a moderate susceptible wheat cultivar Yangmai 158 and the regenerative transgenic plants were obtained (Wang,2009).As a continuous research, the present research mainly focused on the characterization of the function of the Hv-CMPG gene and the molecular mechanism of the powdery mildew resistance mediated by the Hv-CMPG. The results were as followings.1. Blocking ubiquitin-proteasome pathway could lead to increased susceptibility of the powdery mildew resistant wheat variety carrying the Pm21 geneIn vitro ubiquitination assay show that the Hv-CMPG has E3 ligase activity. In order to investigated the relationship of ubiquitination pathway with powdery mildew resistance, the Pm21 carrier Nannong 9918 was treated with MG132, a specific inhibitor of the 26S proteasome, and the DMSO treatments was used negative control. It was found that, compared to the respective control, the occurrence of both the hypha and conidiophore in Nannong 9918 treated for 2h,6h and 12h of MG132 is higher, indicating that the blocking of the 26S proteasome ubiquitination pathway in the resistant variety could increased its susceptibility.2. Transcriptional pattern of Hv-CMPG in different tissues of H. villosa induced by BgtIn leaves, the expression level of Hv-CMPG was up-regulated at 45 min after inoculation, and reached the peak at 1 h after inoculation and then decreased. In stems, the expression of Hv-CMPG was slightly induced, while no significant induced expression was observed in roots.3. Transcriptional pattern of Hv-CMPG in the leaves of H. villosa in response to different hormones and H2O2 treatmentsIn order to characterize the involvement of Hv-CMPG in different signal pathways, the expression pattern of H.villosa in response to SA, JA, ET and H2O2 was analyzed. Two expression peaks of Hv-CMPG were observed at 45 min and 12 h after application of SA. The expression of Hv-CMPG was also up-regulated when treated with ABA and reached the peak at 12 h after inoculation. The expression of Hv-CMPG was maintained at the initial level when treated with JA, ET and H2O2. These suggested that Hv-CMPG involved in the SA and ABA signal pathways.4. Over-expression of Hv-CMPG leads to the accumulation of active oxygen and the cell wall protein cross-linking after inoculation of BgtThe amount of H2O2 accumulation and the cell wall protein cross-linking in the infected leaf epidermal cells were compared using the positive Hv-CMPG overexpression transgenic plants and their receptor variety Yangmai 158. The results showed the amount of H2O2 accumulation in the infected leaf epidermal cells of the Hv-CMPG transgenic plants was significantly higher than that in Yangmai 158 at 12,24 and 48 h post inoculation. As a result, the proportion of secondary hyphae formed in the transgenic plants was lower than that in Yangmai 158.Through the observation of cross-linking of cell wall proteins, it was found that the successfully colonized conidia in the epidermal cells of the transgenic plants with higher density of cell wall protein cross linking were more than that in Yangmai158. It was suggested that Hv-CMPG functioned by blocking the invasion of pathogen into the epidermal cells of the leaves.5. Functional analysis of the Hv-CMPG gene by transformationThe Hv-CMPG gene was transformed into the callus of a moderate susceptible variety Yangmai 158 by a gene bombardment method. Totally,141 regenerated plants were obtained, in which 33 were identified as the positive transgenic plants. The preliminary evaluation result of the plants at the To generation showed that the over expression of Hv-CMPG could increase the powdery mildew resistance of Yangmai 158. |
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