基于pH响应和碳代谢物抑制通路的碳酸铵抑制意大利青霉作用机制研究

Blue mold and green mold were the main diseases of citrus fruits during storage. The extensive application of synthetic fungicides caused great concerns on food safety issues, thus it is urgent to explore green preservation technologies. In our previous research, ammonium carbonate (AC), which is generally regarded as safe and permitted for preservation of organic fruits and vegetables, could effectively inhibit growth of Penicillium italicum and control blue mold of citrus fruits after harvest, and the antifungal activity of the compound was influenced by pH. However, the possible antifungal mechanism involved remains unclear. In this project, mutants with the blockage of pH response pathway, and carbon catabolite repression (CCR) pathway which influence secretion of acids and ammonia and virulence factors will be obtained by gene knockout of pacC and CreA, the key transcription factors of the corresponding pathways respectively; the effects of AC will be investigated on growth, ultrastructural alternation, physiological metabolism and secretion of cell wall degradation enzymes of the wide type strain and mutants; then, with the help of RNA-seq and secreteomic analysis, the effects of AC on gene expressions related to the main biological processes and secretion of virulence factors will be assessed and further verified through real time quantity PCR of the corresponding genes; with the above data obtained, the possible molecular mechanism of AC against the pathogen will be revealed and the interactions with pH response pathway and CCR pathway will be elucidated. In vivo, with the modification of pH and carbon source at the injection sites of citrus fruits inoculated with the strains, the effects of AC on pathogenicity of the wide type stain as well as the mutants will be determined, and the expressions of important genes during colonization will be evaluated, to further verify the possible molecular mechanism involved in the inhibition of the pathogen by AC. The results obtained would provide the basis for further improvement of the disease control efficacy of AC treatment on citrus fruits.

青、绿霉病是柑橘主要采后病害，化学农药引发食品安全问题，迫切需要绿色防腐保鲜技术。我们发现GRAS碳酸铵显著抑制意大利青霉生长并能有效控制果实青霉病，且碳酸铵抑菌作用与改变pH有关，但其作用机制不清楚。本项目拟在前期研究基础上，通过基因敲除技术获得意大利青霉pH响应通路关键转录因子PacC突变株和与pH调控密切相关的碳代谢物抑制通路关键因子CreA突变株；采用生理生化测定与转录组学、分泌蛋白质组学、荧光定量PCR技术分析碳酸铵处理对菌株菌丝生长、生理与代谢特性、pH响应和调控因子表达、致病因子分泌等方面的影响，揭示碳酸铵通过影响pH响应和调控系统抑制意大利青霉生长和致病性的可能作用机制；进一步采用人工接种柑橘果实，通过改变寄主微环境的pH和糖条件，评价碳酸铵对病原菌和突变株活体致病的影响，分析重要基因在侵染进程中的表达，进一步明确碳酸盐抑菌防腐分子机制，为改进该处理控制柑橘病害奠定基础。

青霉绿霉病使柑橘主要采后病害，化学农药引发食品安全问题，迫切需要绿色防腐保鲜技术。前期发现通常认为安全的碳酸铵能显著抑制意大利青霉生长和果实青霉病。其作用机制不清楚。本项目评价碳酸铵与其他碳酸盐对柑橘采后三种主要病原菌意大利青霉、指状青霉和酸腐菌的影响，明确碳酸铵抑制效果最佳，且能有效降低接种病菌的果实菌斑直径。进一步分析碳酸铵抑菌作用离子效应，确定碱性pH、氨释放和碳酸根离子共同构成对意大利青霉抑菌功效。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，明确其作用位点；评价碳酸铵对线粒体呼吸特性、线粒体复合物活性及线粒体超微结构的影响，借助BN-PAGE电泳结合活性染色技术，评价了碳酸铵对线粒体复合物作用特点；采用荧光指示剂和活性氧原位染色技术，揭示碳酸铵引发的活性氧损伤，跨膜电动势、线粒体钙释放、通透性开放孔的影响，分析碳酸铵对菌丝分泌细胞壁水解酶、分泌酸和分泌蛋白的抑制作用，探讨碳酸铵作用机制；利用基因敲除技术获得碳代谢物抑制通路关键因子△CreA突变株，沉默技术获得pH 响应通路关键因子SipacC，△CreA/ SipacC沉默株，明确△CreA突变株、SipacC，△CreA/SipacC沉默株特性，突变株耐碱胁迫严重受损、菌丝胞外酸化能力削弱，抑制细胞比降解酶活性和相关基因表达，基本丧失了对柑橘果实的侵染力。运用转录组学分析，明确△CreA敲除菌株致病力下降的作用机制。探讨碳酸铵对柑橘保鲜效果，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）明确了碳酸铵发挥抑菌作用的离子效应;（2)明确了碳酸铵对意大利青霉呼吸代谢和线粒体功能和超微结构的影响，确定碳酸铵呼吸作用抑制主要位点；（3）获得△CreA突变株、SipacC，△CreA/SipacC沉默株，明确两基因协同调控意大利青霉耐碱与逆境能力和果实致病力（4）运用转录组分析明确了突变株致病力下降的作用机制；（5）探讨了碳酸铵对柑橘果实的保鲜效果。