RLP介导的免疫途径中snc2定点突变基因在棉花抗黄萎病中的功能研究

Verticillium Wilt is one of vascular disease, and it seriously affects the quantity and quality of cotton. SNC2 (suppressor of npr1-1, constitutive 2) gene, encoding a Receptor-Like Protein (RLPs), is a unique system to perform genetic analysis of RLP-mediated resistance pathways. It is different from R-gene-mediated disease-resistant which being wildly studied at domestic and abroad. Previous study found that cotton snc2 transmembrane site mutation gene over-expressed in tobacco, which constitutively activates defense response, and the disease resistance of transgenic tobacco lines were significantly enhanced. It is proved that the gene is resistant to disease. In this study, the GhSNC2 and Ghsnc2 Site mutation gene had been transformed into different cotton materials in northern and southern of Xinjiang, and transgenic materials inoculated with Verticillium dahliae V991, and then carry out the following studies : (1)To study the differences in plant phenotype, gene expression, defense enzyme activity, cell structure and other aspects of transgenic plants and Control, and to reveal the resistance of Ghsnc2 site-directed mutant gene to upcotton under biological stress.(2)To study the response of transgenic plants to pathogen invasion, the relationship between lignin and disease resistance and the relationship between plant phenotype and photosynthesis. Simultaneously we will identify the targeting sites of GhSNC2 gene by constructed fusion vector and analyze the interactions of plants and pathogene by VIGS (virus-induced gene silencing). Our research will explore the mechanism of disease resistance and pathogenesis of the gene from various aspects and angles, and will provide the theoretical basis for the future development of the new cotton germplasm resources of Verticillium dahliae in Xinjiang

棉花黄萎病属维管束病害，严重影响棉花产量和品质。SNC2基因编码类受体蛋白（RLPs），是RLPs介导的抗性途径遗传分析的唯一体系，前期研究发现棉花snc2跨膜区定点突变基因转化烟草，能激活植物的抗性反应，其转基因后代抗病性明显增强；本研究拟对已获得的转化GhSNC2和Ghsnc2定点突变基因的新疆南北疆不同转基因后代棉花材料接种大丽轮枝菌V991，开展以下研究：（1）研究两种转基因植株与受体的抗病性、基因表达、防御酶活性、细胞结构等方面的差异，揭示GhSNC2及Ghsnc2基因在陆地棉受到病原菌胁迫时的抗性反应；（2）研究转基因植株对病原菌侵入的响应、木质素与抗病性关系及植株表型与光合作用的关系。并构建融合载体，探明GhSNC2基因作用位置，利用VIGS，解析基因与病原菌互作关系。从多方面、多角度探索该基因的抗病机理及抗病途径，为今后利用该基因开发新的新疆抗黄萎病棉花种质资源提供理论依据

棉花黄萎病属维管束病害，严重影响棉花产量和品质。SNC2基因编码类受体蛋白（RLPs） ，是RLPs介导的抗性途径遗传分析的唯一体系，前期研究发现棉花snc2跨膜区定点突变基因转化烟草，能激活植物的抗性反应，其转基因后代抗病性明显增强；本研究发现SNC2基因在根、侧根、茎、叶和花中有表达，在苞片中少量表达，且在茎中的表达量最高，说明该基因主要在茎中发挥作用，GhSNC2在烟草中的亚细胞定位实验结果显示在细胞膜及细胞核内均有表达。不同抗病性的棉花受到病原菌诱导后，基因的表达量存在差异。VIGS实验表明GhSNC2基因表达量下降90%后，受到病原菌感染后植株抗病性降低。对已获得的转化GhSNC2和Ghsnc2定点突变基因的新疆南北疆不同转基因后代棉花材料接种大丽轮枝菌V991，发现转定点突变Ghsnc2基因棉花相对抗病效果达到40以上，表现出比其他两者高的抗病性；防御酶POD、PPO、CAT和PAL活性与植株的抗病性呈正相关，其茎部石蜡切片显示皮层细胞排列紧密，细胞间隙很小；GhSNC2基因和病程相关蛋白基因PR-2、PR-3和PR-10均受诱导表达，转Ghsnc2基因植株的病程相关蛋白基因PR-2的表达量升高3-5倍，显著高于其他两者，表明过表达Ghsnc2基因可促进棉花PR基因的表达，激活抗病反应，从而提高棉花对病原菌的抗性；转snc2基因植株更容易利用弱光进行光合作用，病原菌侵染后3-7天转snc2基因植株高于转SNC2基因植株的净光合速率。本研究从多方面、多角度探索该基因的抗病机理及抗病途径，并挖掘调控该基因的miRNAs，为今后利用该基因开发新的新疆抗黄萎病棉花种质资源提供理论依据。