【作者】 赵合情；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2010， 硕士

【摘要】 葡萄是全世界重要的经济水果,葡萄白粉病的发生严重危害其正常生长和代谢。中国野生葡萄包含众多抗病种质资源。本实验室前期从高抗白粉病的中国野生华东葡萄白河-35-1中获得一条抗白粉病新基因乙二醛氧化酶基因(Vitis pseudoreticulata glyoxal oxidase gene, VpGLOX),随后对其进行生物信息学分析、原核表达、抗体制备的研究。本研究就是在此基础上,通过semi-quantitative RT-PCR、Real Time PCR、Western Blot、胶体金免疫电镜、瞬时表达、组织化学染色等技术分析并验证VpGLOX的功能。主要取得的结果如下:1.白粉菌诱导后,VpGLOX在mRNA和蛋白水平的表达分析接种白粉菌不同时间段的不同感、抗葡萄株系或品种中,Real Time PCR分析VpGLOX在转录水平呈现有规律的表达变化,基本呈现先逐渐上调而后逐渐下调的表达趋势,并且在抗病株系白河-35-1的变化趋势远远强于感病的广西-2、佳利酿、无核白。但是,在Western Blot分析的翻译水平上,VpGLOX的表达变化没有转录水平明显,仅仅发生微弱波动。2. VpGLOX表达产物的亚细胞定位分析胶体金免疫电镜定位研究表明VpGLOX蛋白主要定位于叶片组织细胞壁上,在细胞质、液泡等没有分布;并且定位于叶片表皮细胞细胞壁的VpGLOX蛋白明显多于定位于叶片叶肉细胞细胞壁上的。3.瞬时表达研究表明VpGLOX具有产生H2O2的功能在白河-35-1和广西-2的叶片中,瞬时转化pWR306、pWR-VpGLOX后,进行H2O2的组织化学染色和H2O2的含量测定。结果表明,H2O2含量在瞬时转化pWR-VpGLOX的叶片明显高于瞬时转化pWR306空载体的叶片。这说明VpGLOX具有产生H2O2的功能。4. SA、MeJA、Et处理后,VpGLOX的表达分析SA、MeJA、Et分别处理白河-35-1叶片后,通过Real Time PCR检测VpGLOX的表达变化。结果表明,三种处理均引起VpGLOX的表达变化,呈现有规律的表达趋势。MeJA处理后,VpGLOX的表达变化明显,相对表达量呈现先逐渐上调后逐渐下调的趋势,只是在处理3 h时,有一峰值,并在处理24 h时达到最高;SA、Et处理后,VpGLOX的表达变化没有MeJA处理后明显。SA处理后,VpGLOX相对表达量也呈现先上调后下调的趋势,在处理12 h和48 h均有峰值出现。Et处理后,VpGLOX的相对表达量呈现逐渐上调的趋势,只是在处理24 h出现波谷。上述结果说明VpGLOX的表达受信号分子SA、MeJA、Et诱导,VpGLOX可能参与了SA、MeJA、Et的体内合成和SA、MeJA、Et介导的信号传导途径。5. VpGLOX在葡萄不同组织或器官的表达情况分别提取白河-35-1根、茎、叶、卷须和佳利酿根、茎、叶、花、果实、种子的总RNA,通过半定量RT-PCR检测VpGLOX的表达情况。结果表明,VpGLOX在白河-35-1根、茎、叶、卷须中均有不同程度的表达,叶中最强,根中次之,卷须中最弱;在佳利酿根、茎、叶、花、果实、种子中也呈不同程度的表达,花中最强,果实其次,种子再次,根、茎、叶中最弱。这说明VpGLOX在葡萄不同组织或器官的表达没有明显的组织器官特异性,属于组成型表达;生殖器官中VpGLOX的表达明显强于营养器官,推测VpGLOX具有更复杂的功能,可能在生殖器官的生长发育过程中具有更多的防御功能,这些推测仍需要进一步实验来验证。更多还原

【Abstract】 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.更多还原