禾谷镰刀菌环形RNA circ_2518调控DON毒素生物合成的分子机制研究

Deoxynivalenol (DON) is one of the most seriously contaminated mycotoxins in wheat and other cereals, which is mainly produced by Fusarium graminearum. The control of mycotoxins has become an urgent problem in food safety area. Previously, we sequenced and analyzed the noncoding RNA of Fusarium graminearum, and screened 12 candidate circular RNAs related to DON synthesis. Gene knockout and RNA interference experiments showed that circular RNA circ_2518 was involved in the biosynthesis of DON and played an important role in the process of DON production. The program takes circ_2518 as the research object, and gene silencing and overexpression mutants of circ_2518 will be constructed. The function of circ_2518 will be studied. Downstream molecules specifically regulated by circ_2518 under DON production condition will be screened by RIP experiments and high-throughput sequencing technique. The regulated downstream molecules will be confirmed using luciferase reporting system, gene knockout, quantitative PCR and other techniques. The effect of circ_2518 downstream genes and pathways on DON production will be further studied, and its association with TRI genes and cAMP-PKA pathway will be clarified. The molecular mechanism of DON synthesis regulated by circ_2518 will be revealed.

脱氧雪腐镰刀菌烯醇（DON）是小麦等谷物中污染最严重的真菌毒素之一，其主要由禾谷镰刀菌（Fusarium graminearum）产生。控制DON毒素污染已成为食品安全领域迫切需要解决的问题。申请人前期对禾谷镰刀菌的非编码RNA进行了分析，筛选到12个与DON合成相关的候选环形RNA。基因敲除和RNA干扰实验表明，环形RNA circ_2518参与了DON的生物合成，并在产毒过程中起到重要作用。本项目以circ_2518为研究对象，构建circ_2518过表达和基因沉默突变体，开展其功能研究；通过RIP实验和高通量测序筛选产毒条件下受circ_2518特异性调控的下游分子，并利用荧光素酶报告系统、基因敲除、定量PCR等技术进行验证；深入研究circ_2518下游基因或通路对产毒的影响，明确其与TRI基因和cAMP-PKA通路的关联，揭示circ_2518调控DON合成的分子机制。

脱氧雪腐镰刀菌烯醇（DON）是小麦等谷物中污染最严重的真菌毒素之一，其主要由禾谷镰刀菌（Fusarium graminearum）产生。控制DON毒素污染已成为食品安全领域迫切需要解决的问题。本项目对禾谷镰刀菌产毒条件下的非编码RNA进行了分析，筛选到12个与DON合成相关的候选环形RNA。基因敲除和RNA干扰实验表明，环形RNA circ_2518参与了DON的生物合成，并在产毒过程中起到重要作用。项目以circ_2518为研究对象，分别构建了circ_2518过表达和基因沉默突变体及其来源基因缺失突变体等材料，对突变体的表型进行了分析，揭示了其生物学功能；通过RIP实验和高通量测序、蛋白质组等技术对受circ_2518调控的下游分子进行了研究，筛选鉴定到CFEM家族、氨代谢相关基因（GATA家族）和FGSG_01403等关键基因；利用荧光素酶报告系统、基因敲除、定量PCR等技术对筛选到的基因进行了功能分析和系统验证；结合产毒诱导培养和LC-MS/MS分析，深入研究了circ_2518下游基因对产毒的影响，结果表明circ_2518下游分子可以通过氨代谢等路径影响禾谷镰刀菌Tri基因的表达，最终影响DON毒素的生物合成，初步揭示了circ_2518调控DON合成的分子机制。