猕猴桃WRKY转录因子在BTH诱导的溃疡病抗性中的功能和机制研究

China is the world largest production area of kiwifruit. Bacterial canker of kiwifruit has resulted in serious damage to kiwifruit industry. Benzothiadiazole (BTH) is a functional analog of salicylic acid and one of the most effective plant activators that protect kiwifruit from bacterial canker, but the mechanisms underlying its action remain to be investigated. In this project, we are going to study the function and mechanism of the AchWRKY5 and AchWRKY61 in BTH-inducible canker resistance. In this study, we will clarify their characteristics of a transcription factor through subcellular localization and transactivation activity assay. Transgenic kiwifruit plants with enhanced or reduced expression levels of AchWRKY5/61 will be created through overexpression, RNA interference, and CRISPR/Cas9 technology. The physiological and biochemical performance of these transgenic plants after BTH and Pseudomonas syringae pv. actinidiae (Psa) treatment will be evaluated to unravel the function of AchWRKY5/61 in BTH-inducible canker resistance. In addition, AchWRKY5/61-regulated genes that mediate BTH-induced defense responses in kiwifruit will be identified by transcriptome sequencing. Then the mechanism of AchWRKY5/61 in BTH-inducible canker resistance will be preliminarily clarified. This project will provide important theoretical basis for the elucidation of the disease resistance mechanism and the genetic improvement of disease resistance in kiwifruit.

我国是全球猕猴桃最大生产国。猕猴桃溃疡病是威胁猕猴桃生产的毁灭性病害。苯并噻二唑（BTH）是水杨酸的功能类似物，可有效防治溃疡病，但其作用机制尚不清楚。本项目拟对猕猴桃转录因子AchWRKY5和AchWRKY61在BTH诱导的溃疡病抗性中的功能和机制进行研究。本研究将通过体外亚细胞定位和转录活性检测明确其转录因子功能；通过在红阳猕猴桃中超表达或敲除（低）表达AchWRKY5/61基因，分析不同材料在BTH和丁香假单胞杆菌猕猴桃致病变种(Psa)处理下的生理生化表型，解析AchWRKY5/61在BTH诱导的溃疡病抗性中的功能；通过对BTH处理及未处理材料的转录组测序分析获得对BTH响应同时受AchWRKY5/61调控的基因信息，初步阐明AchWRKY5/61在BTH诱导的溃疡病抗性中的机制。本研究结果为猕猴桃抗病机制的阐明和抗病遗传改良提供重要理论依据。

猕猴桃溃疡病是一种严重威胁猕猴桃生产的毁灭性病害。苯并噻二唑（BTH）是水杨酸的功能类似物，可有效防治溃疡病，但其作用机制尚不清楚。本项目通过生物信息学分析鉴定猕猴桃WRKY转录因子家族成员，并构建系统进化树。猕猴桃WRKY基因启动子上的顺式作用元件分析结果表明猕猴桃WRKY转录因子广泛参与植物的胁迫反应。猕猴桃WRKY基因对不同处理（BTH、SA和MeJA等）的转录组分析结果显示，112个猕猴桃WRKY基因中，有101个的表达量至少受一种处理显著影响。其中81个基因受SA或者 MeJA显著诱导表达。AchWRKY5和AchWRKY61 是拟南芥AtWRKY54和AtWRKY70的同源基因，其表达受所有处理显著诱导。亚细胞定位实验表明AchWRKY5和AchWRKY61定位于细胞核；双荧光素酶实验结果表明这两个转录因子均具有转录激活功能；猕猴桃中超表达这两个基因均可增强其对Psa的抗病性。酵母单杂交实验表明AchWRKY61可结合AcSARD1启动子，而不能结合AcSARD2启动子。本项目初步阐明AchWRKY5/61在BTH诱导的溃疡病抗性中的功能。