禾谷镰刀菌抗DON毒素自我保护基因的鉴定和功能分析

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating disease of wheat, barley, and other grain cereals worldwide. It not only causes severe yield losses but also contaminates infected grains with harmful mycotoxins such as trichothecene deoxynivalenol (DON), which is inhibitory to protein synthesis in eukaryotes by binding with L3. Although its molecular target is conserved, F. graminearum is highly tolerant against DON. However, like in many other mycotoxin-producing fungi, the molecular mechanism confering self-protection against DON in F. graminearum is not clear. Two genes in the trichothecene biosynthesis clusters, TRI12 and TRI101, have been implicated in providing some degree of DON-tolerance but the tri12 and tri101 mutants still produce DON. Other genes must exist to be responsible for confering self-protection against DON in F. graminearum. In this study, we will first generate the tri12 tri101 tri5 triple mutant and then use the RNA-Seq approach to identify genes induced by exogenous DON treatment. Because DON is inhibitory protein synthesis, cycloheximide treatment will be used as the control. Genes specifically affected by DON will selected for functional characterization. For genes involved in self-protection against DON, the resulting deletion mutants will be hypersensitive to DON. The function of these genes will be further confirmed by expressing them in Saccharomyces cerevisiae and assaying for increase DON-tolerance. Results from this study will elucidate the self-protection mechanism against mycotoxins in F. graminearum and provide candidate genes suitable for generating transgenic plants with improved FHB resistance and reduced DON contamination.

由禾谷镰刀菌引起的赤霉病不仅严重降低小麦、大麦等粮食作物的产量，其产.生的DON毒素还污染农产品，对人畜健康造成巨大威胁。DON毒素抑制真核生物蛋白合成，但禾谷镰刀菌自身却对DON免疫,如同其它产毒菌，其自身抗毒素分子机制尚未阐明，目前仅发现了两个抗DON毒素自我保护基因TRI12和TRI101,但二者并非病原菌自身抗毒素机制的必需基因，还可能存在其它抗毒素自我保护基因。为此，本研究拟构建TRI12,TRI101和毒素合成酶基因TRI5同时缺失的突变体，以该突变体为材料，以蛋白合成抑制剂放线菌酮处理为对照，采用RNA-Seq深度测序技术在全转录组水平上筛选受外源DON处理特异诱导表达的基因，并对筛选到的基因进行功能分析，从中鉴定出禾谷镰刀菌新的抗毒素自我保护基因，并进一步在酵母中验证其抗毒效果。研究结果将会阐明禾谷镰刀菌自身抗毒素分子机理，也为作物转基因抗赤霉病和降低毒素污染提供候选基因。

由禾谷镰刀菌引起的小麦赤霉病不仅严重降低小麦、大麦等粮食作物的产量，其产生的DON 毒素还污染农产品，对人畜健康造成巨大威胁。DON 毒素抑制真核生物蛋白合成，但禾谷镰刀菌自身却对DON毒素免疫。目前已经在禾谷镰刀菌中发现了两个抗DON毒素自我保护基因TRI12 和TRI101，但二者并非病原菌自身抗毒素机制的必需基因，因此禾谷镰刀菌中还存在其它抗毒素自我保护基因。本项目利用分步基因敲除法成功构建了一个不产DON毒素，同时缺失TRI12和TRI101抗毒基因的突变体菌株，并以该菌株为研究材料，通过添加外源DON毒素诱导禾谷镰刀菌中与抗毒素有关的基因表达，采用RNA-Seq转录组测序技术，共鉴定到541个受DON毒素诱导表达的基因。通过与蛋白合成抑制剂放线菌酮处理的RNA-seq样品进行比较，总共筛选到200个受DON毒素特异性诱导表达的基因，这些基因很可能参与了禾谷镰刀菌自身抗DON毒素过程。通过基因功能验证，明确了30个受DON毒素特异诱导表达基因的功能，并鉴定到一个新的抗毒素自我保护基因，该基因可显著提高酵母抗毒素能力。通过基因功能注释，发现N-乙酰转移酶、乙醛脱氢酶和MFS协同转运蛋白超家族的基因在受DON毒素特异性诱导表达基因里显著富集，揭示了禾谷镰刀菌抗DON毒素调控途径。此外，发现许多DON特异性诱导表达基因在病原菌抗逆、有性生殖和侵染致病过程中具有重要调控作用，揭示了DON毒素调控和有性生殖调控之前的联系。本项目研究结果不仅增进了人们对禾谷镰刀菌自身抗毒素分子机理的认识，也为作物转基因抗赤霉病和降低毒素污染提供了候选基因。

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