玉米黄曲霉抗性基因ZmAFs的功能研究

Aspergillus flavus is one of the most serious diseases in corn production. Aflatoxin is one of the most potent naturally occurring mutagens and carcinogens. Aflatoxins contamination in food and feed causes substantial economic losses worldwide each year. Breeding fungus-resistant maize is the most effective and environmentally-friendly strategy. It has been shown that ZmLOX3 plays an important role in regulating the resistance of maize to Aspergillus flavus. However, how ZmLOX3 is regulated is not clear. Our preliminary studies found that ZmAFs interact with ZmLOX3 to regulate the function of ZmLOX3. The Zmafs mutants have reduced resistance of Aspergillus flavus as the Zmlox3 mutant. The objective of this project is to substantiate the working hypothesis that ZmAFs and ZmLOX3 may coordinately regulate maize resistance to A. flavus through physical interaction and to identify the superior alleles of ZmAFs and ZmLOX3 from a collection of elite maize inbred lines. The expected results will not only deepen our understanding of the regulatory mechanizes of maize resistance to A. flavus，but also provide theoretical support and genetic resources for breeding of maize cultivars with increased resistance to Aspergillus flavus.

黄曲霉病是玉米生产过程中最为严重的病害之一。黄曲霉毒素是自然界最危险的剧毒物质之一，为一级致癌物，其控防难度高，控防成本居高不下，造成世界范围内的巨大经济损耗。培育抗性玉米品种是最为行之有效和环保的控防手段。已有研究表明ZmLOX3在调控玉米黄曲霉抗性方面发挥作用，但ZmLOX3如何被调控进而介导玉米产生黄曲霉抗性的机制尚不清楚。我们前期研究发现，ZmAFs与ZmLOX3互作，调控ZmLOX3的功能，ZmAFs的突变体具有与ZmLOX3的突变体一致的黄曲霉抗性减弱表型，暗示二者可能协同调控玉米黄曲霉抗性。本项目将综合生物化学和作物遗传育种等研究手段解析ZmAFs与ZmLOX3互作进而调控玉米黄曲霉抗性的机理，挖掘玉米中ZmAFs和ZmLOX3与黄曲霉抗性相关的优异单倍型。预期研究结果不仅可以推进玉米黄曲霉抗性相关分子机理研究，也可为玉米黄曲霉高抗品种的分子育种提供理论支撑和基因资源。

黄曲霉病是玉米生产过程中最为重要的病害之一，危害玉米的生产、存储和加工，造成巨大的经济损失。本项目利用酵母双杂交、LCI和BiFC实验证明黄曲霉抗性基因ZmLOX3与ZmAFs的互作关系。构建了ZmAFs和ZmLOX3的敲除和过表达转基因材料，确认了ZmAFs具有对黄曲霉和拟轮枝镰孢菌的广谱抗性基因功能。我们对ZmAFs和ZmLOX3进行了表达模式分析，发现3个基因均为组成型表达，且均在种子中具有较高的表达水平，并进一步分析了发育各个时期种子中这3个基因的表达量。我们对ZmAFs和ZmLOX3进行了亚细胞定位分析，发现ZmAF-1a定位于细胞核中，而ZmAF-1b和ZmLOX3具有核和细胞质双定位的特性。我们进一步综合利用遗传学和分子生物学等手段，证明了ZmAF-1a和ZmAF-1b在SUMO化修饰过程中发挥了重要作用，具有SUMO E3连接酶功能。通过体外SUMO化验证实验，证明了ZmLOX3可被SUMO化修饰，探索了ZmLOX3的SUMO化修饰位点。在上述基础上，初步阐明了ZmAFs与ZmLOX3之间的调控机制，为培育玉米高抗新品种提供了理论支撑和基因资源。