华东葡萄抗白粉病VpMYBR1基因表达与功能分析

【目的】从华东葡萄抗白粉病株系‘白河-35-1’中克隆VpMYBR1基因，并进行基因表达与功能分析，为揭示抗白粉病机制提供理论依据。【方法】采用RT-PCR 和RACE技术克隆VpMYBR1 基因cDNA全长序列，并利用半定量和定量RT-PCR技术进行不同器官和不同处理后的表达分析；通过花序浸染法转化拟南芥，对转基因及未转化对照植株抗白粉病鉴定、台盼蓝染色和抗病标记基因表达分析研究基因功能。【结果】VpMYBR1基因cDNA序列全长539 bp，有228 bp的完整开放阅读框，编码75个氨基酸，包含1个Sant/myb结构域，GenBank 登录号为HQ284197；VpMYBR1基因在‘白河-35-1’不同器官中均有表达，且叶中的表达量最强，花序和果实中表达量最弱；VpMYBR1基因的表达在抗白粉病的华东葡萄‘白河-35-1’与感病的‘湖南-1’、抗病的毛葡萄‘商-24’叶片接种白粉菌6 h后达到最大值，而且在‘白河-35-1’中最高，达接种前的28倍。同样，VpMYBR1基因在SA、MeJA处理后的‘白河-35-1’中的表达量明显高于‘商-24’和‘湖南-1’；将VpMYBR1基因导入野生型拟南芥，提高了转基因株系的抗病性；台盼蓝染色和抗病标记基因表达分析表明，该基因可能通过过敏反应（hypersensitive reaction, HR)提高了转基因株系的抗病性。【结论】从华东葡萄克隆了VpMYBR1基因，基因表达及功能分析表明，该基因具有提高转基因植株白粉病抗性的功能。

Expression and Functional Analysis of VpMYBR1 Gene Resistant to Uncinula necator from Vitis pseudoreticulata

【Objective】 The VpMYBR1 gene was cloned from Vitis pseudoreticulata resistance to powdery mildew clone ‘Baihe-35-1’ leaves infected by Uncinula necator, and its expression model and function were analyzed in this study. This study will provide a theoretical basis for revealing the grape powdery mildew resistance mechanism.【Method】VpMYBR1 was isolated by RT-PCR and rapid amplification of cDNA ends (RACE) approaches, and its expression patterns under different treatments and in different tissues were analyzed by using semi-quantitative RT-PCR and real-time quantitative PCR. The plant expression vector of this gene was constructed, and then transformed into Arabidopsis thaliana by Floral Dip method. The disease resistance identification of transgenic plants was analyzed by infecting powdery mildew, trypan blue staining and analyzing the expression of Arabidopsis defense marker genes.【Result】The assembled VpMYBR1 (GenBank ACC no. HQ284197) cDNA sequence was 539 bp in full length including a 228 bp ORF which encodes a polypeptide of 75 amino acids containing a single Myb-like DNA-binding domain. The VpMYBR1 was expressed in leaves, stems, inflorescence, tendril, and fruit of ‘Baihe-35-1’. The expression of VpMYBR1 was strong in leaves and weak in inflorescence and fruit. The VpMYBR1 was induced by U. necator during 6 to 12 hpi in three grapevine genotypes (V. pseudoreticulata clone ‘Baihe-35-1’, ‘Hunan-1’ and V. quinquangularis clone ‘Shang-24’), the maximum induction of VpMYBR1 was the greatest in powdery mildew resistant genotypes ‘Baihe-35-1’, reaching more than 28-fold. In addition, the VpMYBR1 expression of ‘Baihe-35-1’ induced by defence signal molecular (SA and MeJA) was significantly higher than that of ‘Hunan-1’ and ‘Shang-24’. Then the VpMYBR1 gene was transformed into A. thaliana by Floral Dip method to make further study for verifying its function. The results showed that the expression of VpMYBR1 improved the resistance against the powdery mildew by hypersensitive reaction.【Conclusion】 The results of the study indicate that VpMYBR1 is a founding member of a small family of proteins containing a novel Sant/myb domain which is likely to participate in the regulation of the resistance to U. necator. This research has provided a new candidate gene for improving the disease resistance of V. vinifera by transgenic technology.