小麦籽粒中主要隐蔽型脱氧雪腐镰刀菌烯醇毒素（D3G）污染发生的调控机制研究

Masked mycotoxin of DON-3-O-glucoside (D3G) is usually co-occurring with DON and its acetyl conjugates of 3ADON and 15ADON in naturally contaminated with the highest level and frequency, which seriously aroused the quality and safety problems related to wheat grains in China and thus certainly lead to the great challenges for food safety. However, the factors affected production patterns of D3G contaminant and regulatory mechanisms are unknown yet. The applicant and his group have focused on control of mycotoxins contaminated in foods for more than ten years. And specifically as good basis, several candidate genes and characteristic proteins correlated to production of D3G or DON family mycotoxins, were obtained via phenotypic screening in the constructed mutant isolates of Fusarium graminearum. In this proposal, to address the posed questions, the circumstantial elements including incubation time, temperatures, and light time, as well as the inoculation isolates with various pathogenicity and the wheat grains of different varieties with diverse resistances, were first evaluated, with the goal to elucidate the production pattern of D3G contamination. Afterwards, the samples of wheat grains with highly significantly different D3G production were selected by the well-established liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Furthermore, continuous screening and explanation with obvious functionalities, were performed via combinatorial means of gene chip analysis, 2-D electrophoresis plus Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and High Resolution MS (orbi-trap). As expected, some candidate differentially expressed genes, characteristic proteins and small metabolic molecules related to the D3G production were achieved. To be focused, several core genes, characteristic proteins and small molecules for metabolism playing the key role of D3G production would be ultimately identified through the classic approaches of gene knock-out, and Co-Immunoprecipitation (Co-IP), and Nuclear Magnetic Resonance (NMR). As resulted, the regulatory mechanism of D3G as the mainly masked mycotoxin contaminated in wheat grains are unveiled, supporting as valuable theoretical references to subsequent studies on risk assessment and control of all other mycotoxins contaminated in cereal grains concerning food safety.

主要隐蔽型脱氧雪腐镰刀菌烯醇毒素（D3G）与其原型（DON）、衍生型（3/15ADON）一直协同污染小麦籽粒，严重威胁我国小麦质量安全。而目前D3G污染发生的影响因素、调控机制等均不明确。申请人长期从事真菌毒素污染控制研究，前期筛选到少量与D3G、DON等毒素生物合成相关的基因与特征蛋白，并已构建相应产毒突变体库、表型分析等体系。本项目拟首先明确不同外在环境条件、小麦品种、产毒禾谷镰刀菌等对D3G污染发生的影响作用；进一步针对产D3G差异非常显著的样本，采用基因芯片、双向电泳与MALDI-TOF-MS、高分辨质谱（orbi-trap）等继续筛选与D3G产生显著相关的候选差异基因、特征蛋白与代谢小分子，应用基因敲除、免疫共沉淀、核磁共振等进行功能分析验证，最终阐明与小麦籽粒中D3G污染发生的调控机制，为食品中真菌毒素污染控制提供理论基础。

主要隐蔽型脱氧雪腐镰刀菌烯醇毒素（D3G）与其原型（DON）、衍生型（3/15ADON）一直协同污染小麦籽粒，严重威胁我国小麦质量安全。而目前D3G污染发生的影响因素、调控机制等均不明确。为了明确我国小麦中DON和D3G的污染状况和发生规律，本项目在不同品种的小麦籽粒上接种不同产毒型禾谷镰刀菌进行产毒测定，探究了不同环境条件包括温度、水活度对小麦中毒素累积含量的影响。结果表明DON和D3G的累积量因品种而异，高抗品种小麦苏麦3号和望水白的毒素累积含量相对其他中抗或感性品种更低。同时，毒素的积累量受水活度、温度等非生物因素的影响。针对不同品种的小麦籽粒与禾谷镰刀菌F-1互作样本进行转录组测序分析，发现在相同时间内，感性品种中麦66B被禾谷镰刀菌侵染的程度极高，而高抗品种苏麦3号和中抗品种安农0711被侵染的程度相似。然而，相比于高抗品种苏麦3号，安农0711小麦中的多种代谢通路相关的基因表达量较低，其中与MAPK通路和植物抵御反应相关的基因表达显著较低。通过对3株禾谷镰刀菌菌丝体蛋白组进行分析，成功鉴定出80个差异显著的蛋白。针对转录组和蛋白质数据，选取FGSG_03532，FGSG_03846和FGSG_03486进行进行功能验证，发现这几个基因参与DONs毒素生物合成。另外，通过对三个品种小麦籽粒与禾谷镰刀菌共培养的混合样本进行代谢物分析，共鉴定出422个差异代谢物，主要为脂质和类脂分子（36.83%），有机氧化合物（15.58%），有机酸及其衍生物（15.01%），有机异环族化合物（11.33%）以及糖类和多酮类化合物（9.92%）等，参与甘油磷脂代谢、胆碱代谢、植物次级代谢物合成以及ABC转运蛋白合成代谢等多个生物进程。通过结合转录组和代谢组数据，揭示了不同抗性小麦在被禾谷镰刀菌侵染过程中的分子变化及代谢差异，为食品中真菌毒素污染控制提供理论基础。