中国野生刺葡萄抗白腐病WRKY调控因子功能分析

Grape white-rot is one of the most serious disease that cut production. Our research team screened a Vitis davidii strain ( Vitis davidii Foex 'Tangwei1242' ) that highly resistant to white-rot from Chinese wild grape. Two WRKY genes were cloned from cDNA of 'Tangwei1242', named Vdwrky53 and Vdwrky70. The WRKY family are one of the best-characterized classes of plant defense transcription factors and the most important part of SAR. This research project includes the following three aspects. The firstly, the two TFs are verified belongs to the WRKY family by the yeast one-hybrid system. The secondly, the expression of two TFs and PR gene are detected after pathogen and salicylic acid treated. The regulatory to its downstream PR gene are speculated of Vdwrky53and Vdwrky70 based the results. Finally, these two genes are transferred into Arabidopsis thaliana corresponding mutant, the expression of PR gene are detected after pathogen and salicylic acid treated from recombinant A. thaliana. The regulatory of VdWRKY53 and VdWRKY70 on PR genes and resistant pathways are validated. This research will provide us a foundation theory in WRKY family of grape resistant to disease, and accelerate the research of grape.

葡萄白腐病是严重威胁葡萄生产的四大病害之一，本研究小组从中国野生葡萄筛选到了高抗白腐病的刺葡萄株系'塘尾1242'（Vitis davidii Foex'Tangwei1242'），并以其cDNA为PCR模板，获得了两条受白腐病诱导的WRKY基因序列：VdWRKY53和VdWRKY70。基于WRKY转录因子在系统获得性抗性（SAR）中的重要作用，拟从3个方面进行探讨：首先，通过酵母单杂系统验证其属于WRKY家族；然后，检测在葡萄中这两个转录因子对水杨酸及病原菌的响应，及与其下游PR基因的协同作用，推测VdWRKY53和VdWRKY70基因在抗性通路中的作用；最后，把这两个个基因转入拟南芥相应突变体中，检测重组拟南芥中PR基因的表达变化，并用水杨酸诱导突变株，检测PR基因的表达，从而进一步验证这两个基因的作用途径。初步探讨WRKY在葡萄抗病中的作用，为研究葡萄的抗病机制奠定基础。

从抗病种质刺葡萄同源克隆获得VdWRKY53和VdWRKY70基因的编码区和启动子区域，分析其序列特征，并通过转基因拟南芥分别验证了VdWRKY53和VdWRKY70基因的抗病功能，结果显示VdWRKY53基因显著提高了重组拟南芥的抗病性。转录组测序结果表明，在病菌的初期侵染时，抗病种质刺葡萄感受病菌信号的FLS基因便已经启动，负责信号级联放大的MAPK基因、以及负责开启SA和JA信号途径的NPR1基因及重要病程相关基因PAG、PR和调控基因WRKY转录因子基因均作出了反应和表达量的变化，但是感病种质美人指的免疫启动要到36h后才会启动，表明在抗病的的刺葡萄中存在某种快速应激反应的机制，从而能够快速识别并抵御病原菌的侵入。