Cyclo(Phe-Phe)对棒曲霉素合成代谢调控机理的研究

Cyclo（Phe-Phe）was made up of two phenylalanine, which significantly inhibited the accumulation of patulin of Penicillium expansum. This research will explore the biological property of P. expansum after treated with Cyclo（Phe-Phe）, such as: the colony and spore morphology, spore germinstion, and biomass.Detect the accumulation of intermediate product and patulin by the method of HPLC; Simulaneously measure the content of acetyl-CoA , ATP and mitochondrial membrane potential; RNA-Seq and iTRAQ were used to explore the expression differences of protein on the transcriptional and protein level. All these explorations will elucidate the regulation mechanism of patulin synthesis in P. expansumtreated with Cyclo（Phe-Phe） and find new protein regulators. Finally all these works can complement the mechanism underlying the inhibiton of Cyclic dipeptide to patulin biosythesis, provide reference for searching more effective inhibitors of mycotoxins.

Cyclo（Phe-Phe）由两个苯丙氨酸通过肽键环化而成，可以显著抑制扩展靑霉（Penicillium expansum）产棒曲霉素的积累。本项目采用微生物学方法研究经Cyclo（Phe-Phe）处理对P. expansum菌落、孢子形态、孢子萌发和生物量的影响，借助 HPLC测定棒曲霉素合成中间产物及终产物积累的变化；检测乙酰辅酶A、ATP含量及线粒体膜电位的变化；运用RNA-SEQ和iTRAQ等技术在转录和蛋白水平上筛选经Cyclo（Phe-Phe）处理前后P. expansum表达差异显著且与棒曲毒素合成相关的蛋白，探究其生物学功能及在棒曲毒素合成途径的调控作用。以期阐明由Cyclo（Phe-Phe）引发的P. expansum棒曲霉素合成调控机理，并寻找有关棒曲霉素合成途径新的调节因子。这些研究将对环二肽抑制棒曲霉素合成机理做出新的补充，为寻找到更有效的真菌毒素抑制剂提供参考。

Cyclo(D-Phe-D-Phe)由两个苯丙氨酸通过肽键环化而成，可抑制扩展青霉生长和棒曲霉素积累。本项目采用微生物学方法研究经Cyclo(D-Phe-D-Phe)处理对P. expansum菌落、孢子形态、孢子萌发、生物量和致病性等影响，检测能量代谢相关酶活性及ROS积累并结合透射电镜观测膜的完整性；借助 HPLC测定棒曲霉素积累变化；运用RNA-SEQ和DIA等技术在转录和蛋白水平上筛选经Cyclo(D-Phe-D-Phe)处理P. expansum后表达差异显著且与棒曲毒素合成相关基因和蛋白，通过RT-PCR等分子生物学方法探究其生物学功能以及在棒曲霉素合成途径的调控作用。结果显示：Cyclo(D-Phe-D-Phe)明显抑制菌落直径、孢子萌发率、芽管长度和生物量等，呈浓度依赖性且最低抑制浓度(MIC)为0.4 mM；经透射电镜观察发现细胞质内容物泄露、细胞器解体（液泡大量膨胀）且大规模的内质化、细胞壁变薄且轮廓模糊和细胞膜严重破损，电导率、核酸和蛋白释放量和PI染色荧光值均显著上升；检测能量代谢相关酶活性均下降，影响线粒体功能引发ROS积累并最终导致扩展青霉菌体死亡；经Cyclo(D-Phe-D-Phe)处理扩展青霉后HPLC检测发现在低浓度范围内（≤0.04mM）棒曲霉素积累是下降的，在高浓度范围内（>0.04mM）棒曲霉素的积累是促进的，经Cyclo(D-Phe-D-Phe)处理苹果后接种扩展青霉，发现在大于0.16mM浓度下，棒曲霉素的积累是呈现上升的，说明苹果自身的含糖量、pH等因素也会综合影响棒曲霉素的积累；通过RNA-seq和DIA分析Pat基因簇相关Pat基因表达均不受影响（与实时定量数据一致），说明Cyclo(D-Phe-D-Phe)对棒曲霉素的合成并不是直接作用；而是发现Cyclo(D-Phe-D-Phe)很可能通过影响细胞壁、细胞膜结构组分及功能、糖代谢（淀粉和蔗糖）、氨基酸合成等过程中一些关键基因表达（与实时定量结果一致）而间接调控，后续通过基因敲除和回补实验来探究与细胞膜及细胞壁组分、淀粉和蔗糖代谢相关基因的生物学功能，进一步探究是否通过影响细胞膜功能及糖代谢（蔗糖）而调控扩展青霉生长和棒曲霉素的积累。为探索Cyclo(D-Phe-D-Phe)对扩展青霉生长和棒曲霉素合成抑制机制提供精确且有方向性的线索。