葡萄MrRPV1蛋白LRR结构域的功能研究

【目的】明确葡萄霜霉病抗性蛋白(MrRPV1)LRR结构域对葡萄霜霉菌特异性识别的影响,为进一步研究葡萄与霜霉菌间的互作机理打下基础。【方法】将MrRPV1和白粉病抗性蛋白(MrRUN1)两个蛋白的LRR结构域互换,构建重组表达载体后转化至感霜霉病和白粉病的欧亚种西拉葡萄胚性愈伤组织中以获得转基因植株,并通过DNA、RNA分子水平检测和室内接种试验进行抗病性鉴定。【结果】共获得7个RGA10-8LRR阳性转基因(重组了MrRPV1蛋白LRR结构域)株系和6个RGA8-10LRR阳性转基因(重组了MrRUN蛋白LRR结构域)株系。在7个RGA10-8LRR阳性转基因株系中,仅RGA10-8-13阳性植株表现出与MrRPV1转基因苗S8-1一致的坏死斑点,肉眼观察不到白色霜霉状物,但7个阳性转基因系均未表现出明显的白粉病抗性。在6个RGA8-10LRR阳性转基因株系中,RGA8-10-3和RGA8-10-28两个株系表现出对白粉病的抗性,有35%左右的细胞表现出细胞程序性坏死(PCD);RGA8-10LRR转基因苗均不抗霜霉病。【结论】MrRPV1的LRR结构域可能介导对葡萄霜霉菌的特异性识别,而MrRUN1的LRR结构域可能介导对白粉病菌的特异性识别。

Function of LRR domain in grape MrRPV1

Objective]The function of LRR domain of grape downy mildew resistance protein(MrRPV1) in downy mildew pathogen specific recognition was investigated in order to provide basis for studying the interaction mechanism between grape and downy mildew pathogen. Method]The LRR domains of MrRPV1 and powdery mildew resistance protein (Mr-RUN1) were switched and recombinant expression plasmids were constructed in this study. The recombinant expression plasmids were transformed into embryonic callus of Shiraz grape which was susceptible to downy mildew and powdery mildew so as to obtain transgenic plants. Disease resistance of the transgenic plants was identified by DNA and RNA molecular level detection and indoor inoculation test. Result]In total, seven RGA10-8LRR(recombination of LRR domains of Mr-RPV1 ) and six RGA8-10LRR ( recombination of LRR domains of MrRUN1 ) positive transgenic lines were obtained . Among the seven RGA10-8LRR transgenic lines, only RGA10-8-13 line exhibited similar necrotic spots to MrRPV1 transgenic plant S8-1 with no visible downy mildew. Furthermore, all the seven RGA10-8LRR transgenic lines showed no significant powdery mildew resistance. Powdery mildew resistance was observed in two RGA8-10LRR transgenic lines , namely RGA8-10-3 and RGA8-10-28, with programmed cell necrosis(PCD) in 35% cells. All the six RGA8-10LRR transgenic lines showed no downy mildew resistance. Conclusion]LRR domain of MrRPV1 may play a role in specific recognition of grape downy mildew pathogen and LRR domain of MrRUN1 may play a role in specific recognition of pow-dery mildew pathogen.