刺葡萄0943抗白腐病转录因子VdWRKY49的克隆及序列分析

Clone and analysis of a transcription factor gene VdWRKY49 from Vitis davidii 0943 in grape white rot resistance

【目的】获得来源于中国野生抗病种质刺葡萄0943的转录因子Vd WRKY49基因,揭示其序列特征、基因功能与抗葡萄白腐病之间的关系,初步揭示抗病种质抗病机制。【方法】利用同源克隆的方法分别获得刺葡萄0943的转录因子Vd WRKY49基因的编码区和启动子区域,分析其序列特征,通过实时荧光定量检测其对白腐病菌和水杨酸诱导的反应,同时以感病品种欧亚种‘黑比诺’为对照,分析不同种质中的转录因子Vd WRKY49基因序列及表达差异。【结果】来源于中国野生种质刺葡萄0943的Vd WRKY49基因,在其编码区和启动子区域都有抗病基因的序列特征和位点特征,同时也存在不同,在DNA和氨基酸水平上与来源于欧亚种的‘黑比诺’Vv WRKY49有差异,这些差异可能造成了其结构功能的差异;受到葡萄白腐病和水杨酸诱导后,Vd WRKY49基因的表达量明显高于Vv WRKY49。【结论】来源于刺葡萄0943的Vd WRKY49基因受到白腐病菌和水杨酸诱导后表达量和表达模式明显区别来源于‘黑比诺’的Vv WRKY49基因,推测在葡萄抗病途径中转录因子WRKY49具有重要的生物学作用。

【Objective】To analyze the relationship between VdWRKY49 sequence feature,gene function and resistance to grape white-rot,and reveal the molecular regulatory mechanisms of the resistant germplasm. 【Methods】According to the homologous sequences Vv WRKY49,primers were designed and used to clone Vd WRY49 of Vitis davidii 0943. The resistant founction of Vd WRY49 to grape white-rot was confirmed by bioinformatics analysis and real-time quantitative PCR. The relative expression of WRKY49 of was detected by real-time PCR in V. davidii 0943 and‘Pinot Noir＇after white rot fungi inoculation and salicylic acid treatment.【Results】Some resistance gene features were found at the transcribed region and promoter region of VdWRKY49 from Chinese wild grape V. davidii 0943. In addition,the nucleic acid and animo acid sequence of WRKY49 have differences between V. davidii 0943 and‘Pinot Noir＇,which may cause the changes of protein structure and function. The expression of VdWRKY49 is higher than Vv WRKY49 after treatment with white rot fungi and salicylic acid.【Conclusion】The expression pattern of VdWRKY49 are distinguished from Vv WRKY49 obviously,indicating that VdWRKY49 plays an importantbiological function in grape disease resistance.