Analysis Of The Function And Expression Of A Novel Glyoxal Oxidase Gene Of The Resistance To Uncinula Necator From Chinese Wild Vitis Pseudoreticulata W.T. Wang

Grape is the most economically important fruit of worldwide. Powdery mildew caused by Uncinula necator (Schw.) Burr., leads to significant losses in normal growth and metabolish. Chinese wild vitis contains a number of resistant germplasm. In our laboratory, the novel VpGLOX (Vitis pseudoreticulata glyoxal oxidase) gene was isolated from the Chinese wild Vitis pseudoreticulata accession Baihe-35-1, which is highly resistant to U. necator. Then study with VpGLOX by bioinformatics analysis, prokaryotic expression and antibody preparation in the previous studies. Based on these, to further investigate the function of the VpGLOX gene, semi-quantitative RT-PCR, Real Time PCR, Western Blot, immunogold electron microscopy, transient expression, histochemical detection, and so on were performed. The mainly results as following:1. Expression analysis of VpGLOX in mRNA and protein leavel after inoculation with U. necatorThe expression of VpGLOX at transcriptional level showed a certain pattern by Real Time PCR analysis after inoculation with U. necator in different resistant and susceptible grape accessions or cultivars. Firstly, up-regulated gradually and then down-regulated gradually generally. Secondly, VpGLOX expression at the transcriptional level changed more significantly in the disease-resistant V. pseudoreticulata accession Baihe-35-1 than in the susceptible V. pseudoreticulata accession Guangxi-2, V. vinifera cv. Carignane, and V. vinifera cv. Thompson Seedless. However, the expression of VpGLOX at translational level did not change as significantly as the transcriptional level, only up-regulated slightly.2. Subcellular localization analysis of VpGLOX expression productsImmunogold electron microscopy analysis suggested that immunogold particles representing VpGLOX protein were predominantly localized in cell wall of leaves tissues, while no particles were found in the cytosol, vacuole and so on. The amounts of immunogold particles in cell wall of epidermal cells were much higher than that in the cell wall of mesophyll cells.3. The study of transient expression suggested that VpGLOX has function to produce H2O2Transient expression by agro-infiltration of Baihe-35-1 and Guangxi-2 leaves was performed. The empty pWR306 and pWR-VpGLOX constructs were transiently overexpressed in Baihe-35-1 and Guangxi-2 leaves. Then, histochemical detection of H2O2 and determination of H2O2 concentration were performed. The results showed that the production of H2O2 in Baihe-35-1 and Guangxi-2 leaves overexpressed VpGLOX were more than that infiltrated leaves with Agrobacterium harboring the empty vector, which suggested that VpGLOX could produce H2O2.4. Expression analysis of VpGLOX after SA, MeJA, Et treatmentThe expression of VpGLOX at transcriptional level was determined by Real Time PCR analysis after treatment with SA, MeJA, Et in leaves of Baihe-35-1. The results showed that all the three treatments could cause the change of VpGLOX expression, and certain patterns were presented. After treatment with MeJA, VpGLOX transcripts changed significantly, the relative expression of VpGLOX was up-regulated gradually and then down-regulated moderatly, with the first little peak occurring at 3 h, and the highest point at 24 h. VpGLOX transcripts did not change drastically after SA and Et treatment as MeJA treatment. After SA treatment, the relative expression of VpGLOX was also up-regulated gradually and then down-regulated gentally, with two peaks at 3 h and 24 h. After Et treatment, the relative expression of VpGLOX was up-regulated gradually, only bottomed at 24 h. The results suggested that the expression of VpGLOX was regulated by signaling molecules SA, MeJA and Et. VpGLOX may be involved in SA, MeJA and Et in vivo synthesis and SA-, MeJA-, Et-mediated signal transduction pathway.5. Expression analysis of VpGLOX in different tissue or organ of grapeTotal RNA were extracted from root, stem, leaf, and tendril of Baihe-35-1, and root, stem, leaf, flower, berry, and seed of Carignane.Then the expression of VpGLOX was performed by semi-quantitative RT-PCR. The results suggested that the expression of the VpGLOX mRNA was expressed at different level in root, stem, leaf, tendril of Baihe-35-1. The highest level of expression was in leaf, with lower in root, lowest in tendril. The expression of the VpGLOX mRNA was also showed at different levels in root, stem, leaf, flower, berry, and seed of Carignane. The highest level of expression was in flower, with high in berry, lower in seed, and lowest in root, stem, and leaf. All the above showed that the expression of VpGLOX in different tissues or organs of grape has no significant tissue- or organ-specific. There was a constitutive expression of VpGLOX in grape. VpGLOX expression in reproductive organs was significantly stronger than in vegetative organs, speculating that VpGLOX has more complex functions which maybe play more defensive roles during the development of the reproductive organs growth. However, the possible function still needs to be confirmed further.