赤霉菌HXK1基因调控脱氧雪腐镰刀菌烯醇生物合成的分子机制研究

The mycotoxin deoxynivalenol (DON), mainly produced by Fusarium graminearum, is one of the most prevalent mycotoxins contamination in wheat and other cereal crops worldwide. Previously, we have constructed a mutant of HXK1, a gene that enables to influence a variety of biological processes in cells, with a representative strain isolated from Yangtze River area in China by restriction enzyme mediated transformation. The DON production capability of the deletion mutant, Δhxk1, was significantly lower than that of the wild-type strain as control in rice culture. The results indicated that the HXK1 gene associated with DON production in Fusarium graminearum. Drmatic changes were also observed on hyphal morphology, pigment production and growth rate compared with the wild-type strain, as well as the impairment of sugar catabolism. On this basis, further studies in this proposal will be focused on the molecular mechanism of DON biosynthetic pathway in Fusarium graminearum. The roles and biological functions of HXK1 gene in DON biosynthetic pathway will be resolved. Green fluorescent protein will be fused to the C terminus of HXK1 protein in wild type strain 5035 and the Nikon eclipse 90i epifluorescent microscopy will be used to observe and document HXK1-GFP in specific phrase. The time lapse observation will reveal its cellular localization and dynamic changes in the developmental process. We will also observe the fixed and sectioned mutant hypha/conidiaspore by transmission electron microscopy to investigate the fine structure of mutant cells. Meanwhile, protein localization will be revealed by immunoelectron microscopy with polyclonal antibody against HXK1 protein. This will provide insights into the proper location of this protein in the cell. In addition, differentially expressed genes and significantly changed pathways between the mutant and wild type will be unveiled through gene expression profile analysis. Furthermore, the genes involved in the DON biosynthetic pathway that regulated by HXK1 will be identified and verified. Finally as resulted, the functions and related molecular mechanisms of HXK1 gene in DON production will be illustrated comprehensively, which will provide direct evidences for prevention and control of mycotions contamination in cereals and related products.

脱氧雪腐镰刀菌烯醇（DON）是小麦等谷物中污染水平和污染率最高的真菌毒素，其主要由赤霉菌产生。申请人已证明影响细胞内的多种生物过程的基因HKX1参与DON的生物合成，其敲除突变体Δhxk1产生DON的能力显著降低，致病力、菌丝形态等也发生明显变异，还出现糖代谢缺陷。在此基础上，本项目聚焦DON生物合成的分子机制研究，解析赤霉菌HXK1基因在DON生物合成中的作用和生物学功能；构建HXK1-GFP融合蛋白，研究HXK1在细胞周期中的动态过程及作用位点；以抗HXK1蛋白抗体，免疫定位HXK1在细胞超微结构中的结合特征；采用基因芯片解析HXK1基因缺失对基因组转录的影响及方式，鉴定受HXK1基因调控的与DON生物合成相关的基因。这将揭示赤霉菌中HXK1基因调控DON生物合成的分子机制，直接为谷物及其制品中DON污染的防控提供依据。

脱氧雪腐镰刀菌烯醇（DON）是小麦等谷物中污染水平和污染率最高的真菌毒素，其主要由赤霉菌产生。本项目通过基因敲除获得了赤霉菌HKX1基因缺失突变体Δhxk1。表型分析表明，与野生型菌株相比，突变体Δhxk1产生DON的能力显著降低，且还表现出糖代谢缺陷。此外，Δhxk1接种小麦穗和玉米秆的致病力也显著下降。这些结果表明赤霉菌HXK1基因参与调控DON毒素的生物合成，而且与致病力有关。本项目聚焦DON生物合成的分子机制研究，对赤霉菌HXK1基因进行了功能研究，分析了赤霉菌HXK1基因在DON生物合成中的作用；构建了HXK1-GFP融合蛋白，研究了HXK1在细胞周期中的动态过程及作用位点；分析了HXK1的细胞定位和在细胞超微结构中的分布特点；采用基因芯片分析了HXK1基因缺失对基因组转录的影响，鉴定了一些受HXK1基因调控的可能与DON生物合成相关的基因。