棉花受体蛋白基因家族的抗黄萎病功能解析及其抗病机制分析

Previous studies found that ve1 homologous cotton receptor like genes play a key role in Verticillium resistance. However, only few genes from this family have been studied.This study will use the transgenic Arabidopsis and N. benthamiana with 6 different receptor like genes (Ve1 homologous ones) as materials, and elucidate their disease resistance by inoculating with 15 kinds of Verticillium dahliae. The resistance spectrum of each gene and the different resistance to the same strains would be elucidated. VIGS vectors were generated according to the specific sequences of each gene and each gene of the family can be silenced specifically in island cotton and the resistance of these plants will be examined to further determine the resistant function of each gene. In addition, the key LRR domain will be identified by replacing different LRR domains of Verticillium wilt resistance Gbve1 protein with non-resistance Gbvdr1, which would be important for achieving multi-resistant and broad spectrum resistance by integrating key domains of different resistance proteins. Finally, the transient expression will be carried out to determine the changes of subcellular localization after inoculation of Verticillium wilt by transforming GFP fusion genes with the six resistance one to Arabidopsis and tobacco plants. Reactive oxygen species, apoptosis and callose production will be analyzed after Verticillium wilt infection. By carrying out these experiments, the resistant function and molecular mechanisms of each gene will be identified, and the promising resistance genes may be obtained to cultivate new disease-resistant germplasm.

前期研究发现ve1同源的棉花受体蛋白基因家族是抗黄萎病的关键基因。然而，目前只有该家族中的个别基因对2个菌系的抗性研究报道。为了系统解析这一家族各个基因的抗性谱和抗性机制，本研究拟以该家族6个基因的转基因拟南芥和本氏烟为材料，分别接种15种黄萎病菌系，确定每个基因的抗性谱。根据各个基因的特异序列构建VIGS载体，通过沉默后抗性变化进一步确定这些基因的抗病作用。将筛选到的抗菌谱广、抗性水平高的基因转化棉花。将Gbve1和Gbvdr1基因之间多个LRR区域进行替换，明确抗病蛋白识别病原菌的关键LRR结构域。最后，将这6个基因分别与GFP融合，研究该基因家族的亚细胞定位及在黄萎病侵染前后的位置变化，并检测转基因植株接种黄萎病后的活性氧、细胞凋亡和胼胝质的产生情况。本项目的实施将明确各个基因的抗性作用、阐明这些基因的抗病分子机制，获得抗性谱广、抗性水平好的基因，用于培育抗病新种质。

ve1同源的棉花受体蛋白基因家族是抗黄萎病的关键基因。为了系统解析这一家族各个基因的抗性谱和抗性机制，本研究以该家族6个基因的转基因拟南芥为材料，分别接种15种黄萎病菌系，确定每个基因的抗性谱。结果表明Gbve1, Gbvdr5对落叶型和非落叶型黄萎病菌都具有抗性，而Gbvdr3仅对落叶型黄萎病菌具有抗性；相反，Gbvdr1和Gbvdr6仅对非落叶型黄萎病菌具有抗性。通过农杆菌介导法，将Gbve1和Gbvdr5分别转化棉花，获得23个转基因棉花株系。对目的基因表达量最高的转基因株系Gbve1-1, Gbve1-6以及Gbvdr5-1，Gbvdr5-2进行抗病性鉴定，结果表明该4个株系对落叶型和非落叶型菌株V991和BP2的抗性显著提高。Gbvdr1为无黄萎病抗性的Gbve1的类似基因，将Gbve1的LRR1-6、LRR1-12、LRR1-18、LRR1-24、LRR1-29、LRR1-33替换Gbvdr1相应的LRR重复单元。将替换后形成融合基因构建7个过表达载体，转化拟南芥，对转基因后代分别接种落叶和非落叶型黄萎病菌系V991和BP2,抗性鉴定结果表明，Gbve1的结构域1-6为对V991抗性关键结构域，Gbve1的结构域11-18为对BP2菌株抗性的关键结构域。结构域替换的结果证明棉花抗黄萎病基因的不同结构域对不同菌株抗性具有差异，而不同结构域介导对不同菌株的识别。其创新点在于首次发现棉花抗黄萎病基因不同结构域在识别病原菌不同菌株的差别。根据棉花表面受体蛋白基因在抗感材料中的序列差异，开发出功能基因标记Gbvds1549，其在所有感病材料中扩增出长度为1549bp的片段，在抗病材料中没有扩增产物。通过该功能标记，可以准确检测不同棉花品种或材料的黄萎病抗性，应用于筛选棉花杂交转育后代植株，提高选育效率，显著节约育种筛选成本。最后，将获得的Gbvdr3，Gbvdr5，Gbvdr6基因过量植株接种黄萎病菌株，观察PR基因的表达、活性氧的产生以及胼胝质的积累。结果表明这3个基因过表达后可提高PR基因的表达、促进活性氧的产生以及胼胝质的积累。