植物防御素与作物病原真菌相互作用及其突变分析

The biological pesticide with less-persistent and high efficiency is becoming the research hotspot in field of crop fungal diseases prevention and control due to disadvantages of the chemical pesticide such as high residue and environment pollution. Plant defensin, the main components of the plant immune system, can inhibit the growth of a variety of pathogenic fungus and is expected to develop a new type of antifungal agents.In this project, a novel plant defensin WsAFP2 purified and indentified from wisteria seeds will be used as research material and the biochemical analysis technology such as enzyme-linked immunoassay be employed to indentify the receptor which on sclerotina sclerotium cell membrane interact with WsAFP2. Then, we plan to determine the WsAFP2 and receptor complex three-dimensional structure by nuclear magnetic resonance and to analysis the dynamical process of WsAFP2 interact with receptor with the optimal conformation and kinetic parameters using NMR relaxation experiments and molecular simulation technology. On this basis, WsAFP2 mutation strategy will be established and point mutation and antifungal activities assay be used to screen the antifungal peptide mutants with IC50 achieving to nanomole level base on the primary structure of WsAFP2. This project will lay the foundation for research and development of biological pesticide with less-persistent and high efficiency.

由于化学农药易残留，对环境污染大，因此研究开发高效低残留的生物抗真菌剂是防治作物真菌病害的重要研究方向之一。植物防御素是植物内免疫系统的主要成分，能抑制多种病原真菌生长，有望发展成一种新型的抗真菌剂。本项目以我们课题组从紫藤种子中分离纯化并鉴定的一个新的植物防御素WsAFP2为材料，利用酶联免疫分析等生化分析技术，研究其与油菜菌核病菌细胞膜上鞘脂的相互作用，确定WsAFP2在油菜菌核病菌细胞膜上的作用受体；并应用核磁共振解析WsAFP2和受体复合物的三维结构，结合NMR弛豫实验和分子模拟技术，分析WsAFP2与受体相互作用的动力学过程，明确WsAFP2与受体相互作用的最优构象和动力学参数；在此基础上，建立WsAFP2的突变策略，并利用定点突变技术，结合抑菌活性分析，对WsAFP2的一级结构进行改造，筛选出抑菌活性IC50达到nM级的高效抗真菌突变体，为研究和开发高效低残留生物农药打下基础。

由于化学农药易残留，对环境污染大，因此研究开发高效低残留的生物抗真菌剂是防治作物真菌病害的重要研究方向之一。植物防御素是植物内免疫系统的主要成分，能抑制多种病原真菌生长，有望发展成一种新型的抗真菌剂。本项目以我们课题组从紫藤种子中分离纯化并鉴定的一个新的植物防御素WsAFP2为材料，通过基因工程的方法在大肠杆菌中大量表达重组WsAFP2（rWsAFP2），利用Ni亲和层析等方法纯化得到rWsAFP2。同时利用酶联免疫分析等生化分析技术，确定了WsAFP2在油菜菌核病菌细胞膜上的受体为鞘脂葡萄糖神经酰胺（GlcCer），明确了rWsAFP2对油菜菌核病菌孢子萌发的抑制活性。获得了衍射良好的rWsAFP2蛋白晶体，解析了其晶体结构。在此基础上，建立WsAFP2的突变策略，并利用定点突变技术，结合抑菌活性分析，对WsAFP2的关键氨基酸残基进行了突变，分析了WsAFP2突变体的抑制油菜菌核病菌孢子萌发活性的的影响。为研究和开发高效低残留生物农药打下基础。