海岛棉GbHyPRP1基因抗黄萎病功能鉴定及抗病分子机制初探

Verticillium wilt in cotton caused by Verticillium dahliae is a soil-borne vascular disease, which results in serious loss in yield and fiber quality. Both genetic improvement and research on the resistance mechanism of cotton to V. dahliae are always important scientific problems in breeding and genetics. We have firstly cloned a GbHyPRP1 gene from sea-island cotton, Gossypium barbadense. GbHyPRP1, which take part in resistance of cotton to V. dahliae, is a potentially resistance factor. In order to character and study on the molecular mechanism of GbHyPRP1 involved in resistance of cotton to verticillium wilt, in this project the research is as following: First, we will definitely show the relationship between resistance and HyPRP1 sequence, which contain genes and promoters from different resistant and susceptible cotton varieties; Secondly, we will analyze the expression of HyPRP1 gene and protein in different resistant and susceptible cotton varieties induced with V. dahliae by real-time quantitative PCR and immune electron microscopy; Thirdly, the resistance of GbHyPRP1 to verticillium wilt was character by transformation of upland cotton (Gossypium hirsutum L.) and Arabidopsis thaliana; Fourthly, we will capture GbHyPRP1-interacting proteins in using tandem affinity purification and screen important factor mediated resistance of GbHyPRP1 in cotton proteome, and analyze the value of protein interaction in resistance of cotton to V. dahlia. Project results will not only to provide reliable evidences for research on resistance mechanism of cotton to verticillium wilt, but also have important theoretical and practical significance in molecular breeding for cotton disease resistance.

棉花黄萎病是一种重要的土传真菌维管束病害，严重影响棉花产量和纤维品质。抗黄萎病遗传改良和抗病机制研究一直是棉花遗传育种领域的重大科学问题。项目组首次克隆获得海岛棉GbHyPRP1基因，其参与棉花抗黄萎病过程，是一种潜在的抗病因子。为探究GbHyPRP1抗黄萎病功能与分子机制，本项目拟进行以下研究：一，从不同抗、感棉花品种克隆HyPRP1基因及启动子，明确基因序列差异与品种抗病性的关系；二，采用荧光实时定量PCR和免疫电镜技术，分析黄萎病菌胁迫下基因和蛋白在不同抗、感棉花品种中的表达差异；三，遗传转化陆地棉品种和拟南芥，明确GbHyPRP1基因抗黄萎病功能；四，利用串联亲和纯化技术从棉花蛋白组中捕获GbHyPRP1互作蛋白，筛选介导GbHyPRP1抗病反应的重要因子，分析蛋白互作的抗病意义。项目结果将不仅为棉花抗黄萎病机制研究提供理论依据，还将对棉花抗病分子育种具有重要的理论和应用意义。

棉花黄萎病是一种重要的土传真菌维管束病害，严重影响棉花产量和纤维品质。抗黄萎病遗传改良和抗病机制研究一直是棉花遗传育种领域的重大科学问题。项目组首次克隆获得海岛棉GbHyPRP1基因，其编码蛋白包含富含脯氨酸重复域和花粉过敏原域。GbHyPRP1定位于细胞壁，其在棉花根和茎中有较高的表达，特别是当受到黄萎病菌侵染后发生显著下调表达。GbHyPRP1启动子序列中含有几个植物激素反应元件，进一步的研究证明其受SA诱导发生显著下调表达。我们通过过表达和病毒诱导的基因沉默技术从两个相反的方向对HyPRP1的抗黄萎病功能进行了验证。HyPRP1过表达显著降低了拟南芥对黄萎病的抗性。相反，HyPRP1沉默显著提高了棉花对黄萎病菌的抗性。为了进一步分析HyPRP1介导的棉花抗黄萎病机制，我们利用RNA-seq技术比较了HyPRP1沉默前后整体转录组发生的变化。我们发现在黄萎病菌处理后，HyPRP1沉默棉苗细胞壁发生显著增厚，木质素含量显著增加。此外，HyRPP1沉默能够显著提高ROS在棉花根尖的积累。综上，我们的研究结果表明HyPRP1在棉花抗黄萎病过程中通过加厚细胞壁和提高ROS积累，进而发挥重要的负调控作用。项目结果不仅为棉花抗黄萎病机制研究提供了理论依据，还对棉花抗病分子育种具有重要的理论和应用意义。