黄曲霉细胞凋亡中钙信号调控网络应答紫苏醛的机制研究

Increasing key global food safety concerns has been directed towards food contamination caused by Aspergillus flavus. Management of A. flavus contamination of food has relied heavily on the use of synthetic preservatives so far. As a green and safe compound, perillaldehyde (PAE) showed significant antimicrobial activities. Employing PAE as a significant food preservative during storage is very promising. In our previous studies, it was found that PAE induced apoptosis in A. flavus cells together with an increase of intracellular Ca2+ level, which indicated Ca2+ are involved in apoptosis in A. flavus, however, its mechanism of action has not yet been ascertained. Thus, we will take intracellular calcium signal as the original focus to go on the upstream of apoptosis in A. flavus in the resent project. 1) We set out to detect the response dynamics of calcium signals and apoptosis in A. flavus through techniques such as Non-invasive Micro-test Technique. 2) Confocal laser scanning microscope and flow cytometry were used to examine key markers for PAE-induced apoptosis in A. flavus. 3) We investigated the funtions and relation of the apoptosis upstream gene regulated by calcium signalling and its downstream genes through knock-out and rescue method for interested genes, mRNA and protein analysis of interested genes, protein localization by immunofluorescence. The mechanisms of calcium signalling networks in response to perillaldehyde on the regulation of apoptosis in Aspergillus flavus will be elucidated at the cellular, subcellular and molecular levels. This study will reveal a novel antifungal PAE target from molecular level and provide new technological approaches for the research and development of high efficient and safe food preservatives.

黄曲霉引起的食品污染已成为全球关注的重大食品安全问题。目前主要以化学防腐剂控制黄曲霉引起的食品污染。绿色安全化合物紫苏醛其抗菌活性显著，在食品保藏中应用前景广阔。前期研究发现，紫苏醛可提高黄曲霉胞内钙离子浓度，诱导黄曲霉细胞凋亡。研究提示钙离子可能参与凋亡过程，然而具体作用机制尚不清楚。本项目拟以钙信号为黄曲霉凋亡上游信号切入点，采用1)非损伤微测技术等考察紫苏醛刺激下黄曲霉胞内钙信号对凋亡作用的响应动力学；2)激光共聚焦显微镜、流式细胞仪等检测紫苏醛诱导黄曲霉凋亡的生理生化特征；3)相关基因敲除及互补、基因和蛋白表达检测、蛋白荧光定位等手段研究凋亡上游钙信号调控基因及其下游关键基因的功能并验证基因间的上下游协作关系，从细胞-亚细胞-分子水平研究黄曲霉细胞凋亡中钙信号调控网络应答紫苏醛的机制。研究结果将从分子层面揭示紫苏醛抗真菌作用新靶点，为高效安全的新型食品防腐剂研发提供新技术途径。

黄曲霉（Aspergillus flavus）引起的食品污染已成为全球关注的重大食品安全问题。目前主要以化学防腐剂控制A. flavus引起的食品污染。绿色安全化合物紫苏醛（Perillaldehyde, PAE）其抗菌活性显著，在食品保藏中应用前景广阔。前期研究发现，PAE可提高A. flavus胞内钙离子浓度，诱导A. flavus细胞凋亡。研究提示钙离子可能参与凋亡过程，然而具体作用机制尚不清楚。本项目通过流式细胞术、荧光显微镜、蛋白免疫印迹法、实时荧光定量PCR、非损伤微测技术、电子扫描显微镜和透射电子显微镜等技术检测发现，PAE可诱导A. flavus胞内引起活性氧（Reactive oxygen species, ROS）和钙离子（Ca2+）浓度的升高，导致线粒体膜电位降低，促进凋亡因子细胞色素C从线粒体释放到细胞质中，激活Metacaspase。同时能观察到A. flavus发生磷脂酰丝氨酸外翻、DNA损伤和核碎裂等早期凋亡和晚期凋亡的形态特征。上述研究表明，A. flavus中ROS累积在Metacaspase介导的凋亡中起着关键作用。通过转录组测序分析发现不同浓度PAE处理A. flavus后，具有抗氧化功能的谷胱甘肽代谢通路基因上调，促进ROS清除。但是，PAE能够抑制糖酵解（EMP）和磷酸戊糖途径（PPP）的基因表达，抑制还原力NADH和NADPH的产生，减少ROS清除反应的还原力。同时，PAE还能够抑制MAPK中的过氧化氢酶（catA）的表达，降低ROS的清除过程。通过荧光定量PCR验证了转录组数据的准确性，发现PAE能够促进A. flavus中抗氧化转录因子yap1和skn7的表达，促进还原蛋白glr1和hyr1的表达；抑制糖代谢通路中的脱氢酶（gpdA）表达和MAPK通路中的CatA表达。此外，PAE还能够诱导钙离子/钙调蛋白依赖的蛋白激酶（cmkA）上调表达从而对A. flavus起到抑菌作用。因此，本研究为将PAE开发成新型的食品防腐剂提供理论支持，同时为解决以A. flavus为首的食品腐败问题以保障食品安全具有重要意义。