香菇中活性物质抑制脱氧雪腐镰刀菌烯醇及其衍生物生物合成的分子调控机制

In our previous studies, the bioactive compounds isolated from Lentinula edodes have been proved to efficiently inhibit the biosynthesis of deoxynivalenol and its derivatives. However, until now, these bioactive compounds are unclear, and there are no investigations about the related molecular regulation mechanisms of this inhibition procedure. In the current research proposal, the techniques of microbiology, molecular biology and analytical chemistry will be comprehensively utilized to isolate and identify the bioactive compounds from Lentinula edodes, which could inhibit the biosynthesis of deoxynivalenol and its derivatives. Then, the transcriptomics and proteomics will be combined using to analyze the expression of the differential genes and proteins between the Fusarium spp. treated with bioactive compounds and the control ones, so as to screen the related regulation genes participating in inhibiting the biosynthesis of deoxynivalenol and its derivatives. The function of candidate regulation genes will be further studied by making gene knock-out, knock-out-complemented and over-expressed mutants. After investigation of the production of DON and its derivatives and the expression of key genes (such as Tri genes) in the wide type strains and the mutants inoculated in PDA medium treated or not treated with bioactive compounds isolated from Lentinula edodes, respectively, the molecular regulation mechanisms of inhibition effects of these bioactive compounds on biosynthesis of deoxynivalenol and its derivatives will be preliminarily revealed to provide theoretical foundamention and experimental data for preventing and controlling the contamination of deoxynivalenol and its derivatives in agricultural products, and also, the findings in the present study are valuable for mining another important application field for Lentinula edodes, so as to substantially promote the development of related industries.

前期研究发现，香菇中活性物质可以有效抑制脱氧雪腐镰刀菌烯醇（DON）及其衍生物的生物合成，目前该活性物质的种类不清楚，未见相关抑制过程分子调控机制的研究。本项目拟综合利用微生物学、分子生物学和分析化学等技术手段，分离鉴定香菇中具有抑制DON及其衍生物生物合成作用的活性物质，进而采用转录组和蛋白组等组学技术，综合解析经活性物质处理和未处理的镰刀菌菌株的差异性基因及蛋白表达谱，筛选参与抑制DON及其衍生物生物合成的相关调控基因；构建候选基因的敲除、敲除回补、过表达突变体菌株，通过研究野生型菌株及突变体菌株在有无香菇活性物质处理时DON及其衍生物的产生以及关键基因（如Tri基因）表达情况，最终初步阐明香菇中的活性物质抑制DON及其衍生物生物合成的分子调控机理，为利用香菇中的活性物质防控农产品中DON及其衍生物污染提供理论基础和数据支持，同时，将挖掘香菇的另一重要应用领域，推动相关香菇产业的发展。

脱氧雪腐镰刀菌烯醇（DON）及其乙酰化衍生物（3-ADON和15-ADON）是镰刀菌产生的重要真菌毒素，具有急性毒性和慢性毒性，被确定为最危险的食品污染物之一。前期研究发现香菇提取物可以有效抑制DON及其衍生物的生物合成，但具体活性组分和调控机制还不清楚。本项目首先综合利用中压液相色谱、超高压液相色谱飞行时间质谱、气相色谱和核磁共振波谱等技术对香菇提取物中的活性成分进行追踪、分离和鉴定，发现精氨酸等化合物为香菇提取物中抑制DON及其衍生物生物合成的主要物质。后续的验证实验明确了PDA培养基中10 mM精氨酸等活性物质可以有效抑制镰刀菌产生DON及其衍生物，进而采取RNA-seq分析筛选参与调控的相关基因，发现精氨酸作用下DON合成关键基因TRI101显著下调；同时，对差异表达基因进行GO和KEGG功能富集分析，发现差异表达基因主要涉及糖类、蛋白和代谢小分子等的转运、合成和代谢等生物过程，揭示了精氨酸可能通过干扰禾谷镰刀菌中核糖体功能、过氧化物酶体合成、氨基酸和糖类的合成及代谢等途径从而下调毒素合成关键基因TRI101的表达，最终抑制DON及其衍生物的合成。最后，本项目考察了小麦、玉米等农产品基质中DON及其衍生物的产生规律，并在最适产毒条件下验证精氨酸的抑菌作用，结果显示高浓度精氨酸可以有效抑制小麦、玉米等中禾谷镰刀菌的生长和DON及其衍生物的产生。本项目的研究成果为进一步深入解析DON及其衍生物合成调控机制提供了理论基础，为利用精氨酸防控DON及其衍生物的污染提供了科学依据和技术支撑，从而保障我国的食用农产品质量安全。在本项目的资助下，已发表SCI论文13篇，申请国家专利一项，培养硕士研究生2名。