月季广谱高抗白粉病基因RhMLO1和RhMLO2抗病性的分子机制

Powdery mildew caused by Podosphera pannosa was the most common disease in cut rose production globally. It is also one of the main diseases of garden roses and pot roses. Yunnan is one of the distribution centers of wild roses in China, and it is also one of the main cut rose producing areas in the world. However, the unclear resistance mechanism of wild roses to powdery mildew and the lack of related resistant genes have been limiting the utilization of wild roses in the genetic improvement of modern rose cultivars. Recently, wild types of resistant genes to powdery mildew, RhMLO1 and RhMLO2, have been isolated and their functions have been preliminarily identified by us. Based on this, we are going to illustrate the subcellular position by combining the methods of fusion protein construction, infection, fluorescence microscope observation in this study. After identifying the function of RhMLO1, we will transfer RhMLO2 to roses to analyze its function. In addition, we will select and identify the interacting proteins of the two genes using suitable methods. The results will help to understand the subcellular position, resistant function and interacting proteins of RhMLO1 and RhMLO2. It is also useful for illustrating the molecular mechanism of broad-spectrum resistance of the objective genes, and will supply basic knowledge for the utilization of these disease resistant genes in the genetic engineering of ornamental plants.

由白粉病菌引起的白粉病害是全球切花月季生产的第一大病害，也是庭院月季和盆花月季的主要病害之一。云南是中国蔷薇的分布中心，也是全球切花月季的主产区之一，但对蔷薇野生资源的优异性状/基因缺乏研究制约了其在现代月季遗传改良中的应用。项目组在对已获得的月季野生型基因RhMLO1和RhMLO2进行初步鉴定及功能分析的基础上，拟通过融合蛋白的构建、侵染、荧光显微观察等实验，明确RhMLO1和RhMLO2的亚细胞位置；在明确RhMLO1基因功能的基础上，构建RhMLO2基因的过表达和沉默/反义载体，通过转基因技术验证该基因回到原作物中的功能；利用相关技术筛选和鉴定与RhMLO1和RhMLO2的互作蛋白。以期揭示RhMLO1和RhMLO2的体内作用场所、抗病功能及互作对象，解析目的基因的广谱抗白粉病的分子机理，并为该类基因在观赏植物抗病遗传工程中的应用奠定基础。

白粉病害是切花月季生产的第一大病害。Mlo基因是一类新型的抗病基因。本项目围绕Mlo基因在月季中的抗白粉病机制，探索了该基因的亚细胞位置、功能和互作蛋白等科学问题。分别构建了RhMLO1:GFP和RhMLO2:GFP的融合蛋白，以农杆菌为介导分别侵染植物表皮细胞，借助荧光显微镜观察细胞中的荧光信号的位置，结果表明它们均主要分布于细胞质膜、液泡膜和细胞核，明确了RhMLO1和RhMLO2蛋白的亚细胞位置即功能场所，证实了该蛋白是一类跨膜结构的蛋白；在已完成RhMLO1基因功能验证的基础上，分别构建RhMLO2基因的正义和反义载体，将其分别转化月季植株，获得转基因月季株系。结果表明：正义载体的导入使转基因植株白粉病抗性降低；反义载体的导入使转基因植株白粉病抗性增强，明确了RhMLO2基因的抗病性功能；通过Co-IP和Western Blot等相关实验，初步筛选出2个与目标蛋白RhMLO1和RhMLO2互作的蛋白：transcription factor b3（转录因子B3）和TIR-NBS-LRR resistance protein muRdr1A（TIR-NBS-LRR抵抗蛋白质muRdr1A）。本项目不仅为发掘白粉病抗性基因提供了科学依据，而且项目建立的月季遗传转化体系为培育抗白粉病月季新品种提供了可操作的技术体系和基因。项目发表相关研究论文4篇，其中SCI收录1篇；申请并获得1项国家技术发明专利；项目负责人于2019年入选云南省“万人计划”中的青年拔尖人才培养项目。