设施辣椒中茄腐镰刀菌致伏马毒素污染的发生规律与分子调控机制研究

Greenhouse pepper is highly specialized and economically important agricultural products in China, which also contributes to more and more proportions in the whole dietary consumption.Till now, very few research targeting production pattern and molecular regulatory mechanism of mycotoxins contaminated in greenhouse pepper are performed worldwide, which leads serious menace to food safety issue and related industry development. In our previous studies, Fusarium solani presented as dominant species causing rot disease in greenhouse pepper, within in the population of almost 500 Fusarium isolates originating from various geographic locations in China, where fumonisions occurred as main mycotoxin contaminations there. More interestingly, the Fusarium. solani strains were capable of producing fumonisions in liquid culture, inoculated greenhouse pepper, as well as maize and rice cultures. As summarized, the above mentioned results are obviously different to those previously reported in other countries. In this proposal three Fusarium solani strains with high, medium and low fumonisions-producing capabilities identified via the well-established LC-MS/MS method are first inoculated with greenhouse pepper under different incubation time and conditions (temperatures, light time, and fungicide carbendazim treatment), in order to elucidate the production pattern of fumonisins contamination caused by Fusarium solani in greenhouse. In parallel, the Fusarium solani isolate with the highest fumonisions production capability is selected for full-genome analysis via de novo sequencing and related comparative genomic analysis, in order to unveil the candidate genes and related expression patterns responsible for fumonisions production, Moreover, the separately incoculated samples with the isolates of highly producing and non-producing of fumonisins are prepared intowith halves for protemoic anlysis via 2-D electrophoresis and MALDI-TOF-MS, and metabolic analysis by LC-TOF-MS, HRMS (orbi-trap) and NMR, respectively. More specifically, one or two key genes responsible for fumonisions biosynthesis via RT-PCR and gene-knock out analysis are selectively confirmed. Also, the functions of one or two differential proteins and metabolites of small molecules are individually verified by the approaches of antibody preparation and western blot, chemical synthesis and in vitro evaluation. Finally as resulted, the interaction and molecular regulatory mechanisms of the confirmed elements of genes, proteins and metabolites of small molecules are unveiled to elucidate the production patterns and molecular regulartory mechanisms of fumonisions contaminations caused by Fusarium solani in greenhouse pepper. This would be valuable theoretical references to subsequent studies on risk assessment and control of mycotoxins contaminated in greenhouse vegetables concerning food safety.

设施辣椒是我国特种优势农产品，占膳食消费比重也越来越大。国内外关于其污染真菌毒素的相关基础研究很少，给食品安全及相关产业带来严重隐患。课题组已确定我国设施辣椒中以茄腐镰刀菌为优势菌，以伏马毒素污染为主；且该茄腐镰刀菌在不同基质中均产伏马毒素，与国外报道明显不同。本项目拟采用产毒高、中、低菌株接种辣椒，在不同环境条件下培养，阐明伏马毒素污染的发生规律；针对产毒最高的茄腐镰刀菌进行de novo测序分析，揭示伏马毒素产生的分子基础；采用双向电泳、MALDI-TOF-MS、LC-TOF-MS、高分辨质谱orbi-trap、NMR等挖掘差异蛋白与代谢小分子；采用RT-PCR与构建基因缺失株、抗体制备与western blot、化学合成与体外评估等分别验证1-2个关键基因、差异蛋白与小分子功能。最终揭示设施辣椒中茄腐镰刀菌致伏马毒素污染的发生规律与分子调控机制，为后期相关安全评价与控制奠定理论基础。

设施辣椒是我国特种优势农产品，占膳食消费比重也越来越大。国内关于其真菌毒素污染的相关基础研究很少，给食品安全及相关产业带来严重隐患。伏马毒素是镰刀菌次级代谢产生的一种真菌毒素，可降低农作物的生产性能、损伤动物和人的免疫系统、干扰鞘脂代谢，对人和动物健康构成严重威胁。为了明确我国设施辣椒中伏马毒素的污染状况和发生规律，本实验从全国采集了34个城市地区的设施辣椒样本1000份，通过分离鉴定到45株镰刀菌，隶属8个种，主要有茄腐镰刀菌（F. solani）、木贼镰刀菌（F. equiseti）、多育镰刀菌（F. proliferatum）藤仓镰刀菌（F. fujikuroi）、串珠镰刀菌（F. verticillioides）和尖孢镰刀菌（F. oxysporum）。分别接种大米培养基和设施辣椒上进行产毒测定，并通过LC-MS/MS进行伏马毒素定量分析，结果表明F.solani在21d时伏马毒素总量达到最高，FB1和FB2(FB3)含量分别是683.3 ng/g和192.6ng/g，在21d后毒素含量逐渐下降，并在大米培养基上的产毒水平高于设施辣椒。针对产毒最高的F.solani进行de novo测序分析，在核苷酸水平上，未发现伏马毒素合成相关基因簇，但在蛋白水平上，发现有同源性在40%以上的蛋白与FUM1、FUM6、FUM11、FUM18等相似，同时发现了与伏马毒素合成调控相关的一些基因，如GBP1、CPP1、FST1、GBB1等。结合这些结果，我们选取3-4个同源性高的基因，通过构建敲除突变株来验证。但得到的突变株产毒表型没明显变化。另外，通过蛋白质组比较分析，对在大米培养基上的3株F.solani蛋白组进行分析，共鉴定出512个差异表达蛋白质。选择差异表达达到3倍以上的点进行质谱鉴定，成功鉴定出70个差异显著的蛋白。鉴定的蛋白主要参与了生物合成和小分子代谢、细胞氮成分代谢、碳水化合物代谢和胁迫反应等10个生物进程。这一部分数据目前正在进一步分析中，目前正结合de novo和蛋白质组数据，选出共有的差异基因，进行突变株构建和测定，为系统阐明设施辣椒中F.solani致伏马毒素污染的发生规律与分子调控机制提供了科学依据，为后期相关安全评价与控制奠定理论基础。