基于线粒体功能的蒎烯抑制柑橘青霉菌作用机制研究

Blue mold cause great loss of citrus fruits, the nowadays applied synthetic fungicides, raise great concerns due to the appearance of resistant strains and environmental risks, thus, it is urgent to explore high efficacious, safe biological preservatives as alternative. Preservation with pine needles, was a traditional appoarch for citrus fruits. Previously, we found that pinenes, the major components of pine needle essential oil, demonstrated strong inhibition against Penicillium. italicum. Pinenes could inhibit mecylia respiration, reduce ATP production, increase membrane permeability, induce large vacuole appearance and cell apoptosis, while its anolog, lemonine, showed much weak inhibitory activity. In this project, using limonine as negative compound, we'd like to investigate the effects of pinene on mitochondrial respiratory elctronic transport chain, by application of respiratory inhibitors and activators, and to elucidate the possible action site(s) of pinene, combined with measurements of the activities of the complexes of elctronic transport chain; to evaluate ROS damage on pathogen mitochondria induced by pinenes, with the help of fluorescent indicators for reactive oxygen species production, and analysis of the antioxidant components and protective enzymes as well as thiobarbituric acid reactive substances indicating membrane lipid peroxidation; to explore the effects of active component on mitochondrial permeability transition pore (MPTP) stage which indicates cell apoptosis, with evaluations of calcium release, transmembrane potential and swelling of mitochondria, by application of MPTP enhancers and inhibitors, and to access its effects on components and functions of cell membranes and cell wall. Combined with the above observations we'd elucidate the primary action site(s) and the possible target(s), and the physiological mechanisms of the pathogen induced by pinene, and further prove the action mode of pinenes and the molecular mechanism with RNA sequencing and validations of important genes by quantitive real time polymerase chain reaction, which would provide fundermental bases for the new-type fumigating agent for citrus postharvest disease control

采后柑橘青霉病害发生严重，目前化学防治存在抗性菌株和安全性等问题，寻找安全、高效生物防腐剂成为热点；松针保鲜是柑橘传统保鲜方法，前期发现松针挥发油组分蒎烯强烈抑制青霉，能抑制菌丝呼吸，降低ATP，增加膜透性；诱导菌丝空泡化和细胞凋亡，其结构类似物柠檬烯无抑菌作用。本项目拟以柠檬烯为参照，运用呼吸抑制剂和激活剂，确定蒎烯对真菌呼吸电子传递影响，结合电子传递链相关酶活性测定，初步明确其作用位点；运用荧光指示剂显示活性氧产生，结合抗氧化物质和酶分析及膜脂过氧化测定，评估活性氧对真菌线粒体损伤；分析真菌线粒体钙释放、跨膜电动势、线粒体溶胀等特性，并运用通道促进剂和抑制剂，探讨活性成分对线粒体通透性转换的作用；并评价蒎烯对细胞膜、细胞壁的影响，探明蒎烯的作用部位与作用靶标和相应生理机制；结合RNA-seq转录分析和重点基因定量表达验证，明确蒎烯抑菌作用机制，为柑橘新型防腐剂开发奠定基础。

结题摘要： 由意大利青霉引起的柑橘青霉病是柑橘采后重要病害，研究新型、低毒、高效的生物防腐剂对于柑橘的安全生产具有重要意义，从传统生物保鲜方法中松针的活性成分是拍戏，能有效抑制意大利青霉菌。本项目以意大利青霉为研究对象，评价蒎烯的抑菌活性，确定其最低抑菌浓度；在此基础上评价蒎烯处理对意大利青霉菌丝呼吸代谢的影响，结合呼吸作用抑制剂和呼吸代谢主要酶活性评价，初步明确其作用位点；建立意大利青霉线粒体活性提取方法，评价蒎烯对线粒体复合物活性，线粒体呼吸特性及线粒体超微结构的影响，结合BN-PAGE电泳结合活性染色技术，评价了蒎烯对线粒体复合物作用特点；运用荧光指示剂和活性氧原位染色技术，揭示蒎烯的活性氧损伤，分析其对跨膜电动势、线粒体溶胀、线粒体钙释放、细胞凋亡的影响；评价蒎烯对细胞膜透性和组分、细胞壁组分和酶活性的影响，探明蒎烯的作用部位与相应作用机制；运用转录组分析，明确蒎烯抑菌作用机制；探讨了蒎烯和蒎烯缓释剂对柑橘保鲜的效果，为其生产实际应用奠定了基础。四年来，项目组研究目标和研究内容，基本按照预先设定的技术路线和实验方法，较好的完成了项目任务。经过了大量的实验（详见研究报告正文部分），主要取得以下成果：（1）明确了蒎烯对意大利青霉呼吸代谢和线粒体功能和超微结构的影响，确定了蒎烯对呼吸抑制的主要位点。（2）明确了蒎烯对意大利青霉的细胞膜和细胞壁作用。（3）阐述了蒎烯抑制意大利青霉的作用机制。（4）探讨了蒎烯和蒎烯缓释剂对柑橘果实的保鲜效果。.本项目按照研究计划进行，基本完成研究目标，明确了蒎烯对意大利青霉抑菌作用的组织结构改变、生理代谢损伤、分子作用机制，归纳出蒎烯主要抑制呼吸和线粒体功能损伤，导致活性氧积累和细胞凋亡，并显著影响膜脂组分代谢和细胞壁代谢，从而有效抑制意大利青霉作用。项目累计发表SCI论文2篇，EI期刊2篇，核心期刊1篇。项目目前仍有数据和内容在整理中，预计将发表1-2篇论文。