基于稳定同位素示踪的“拟”靶向代谢组学方法研究发菜响应高盐胁迫的代谢调控机制

Nostoc flagelliforme (N. flagelliforme) is one of the terrestrial nitrogen-fixing cyanophytes with important resource value and ecological value, mainly grown in the desert and poor soil. Environmental factors are the inevitable stress factors during the growth process. However, the studies on the molecular mechanism of N. flagelliforme in response to the extreme environment stress are very limited, and the metabolic regulation mechanism based on small molecule compounds has not been carried out as far as we know. In the previous study, we preliminarily established a novel pseudotargeted metabolomics mehtod of N. flagelliforme based on GC SIM/MS and UPLC MRM/MS. Based on this, we intend to further establish a stable isotope-assisted pseudotargeted metabolomics approach, then to study the time-course variations of metabolite concentration and dynamic transformation process of isotope carbon source of N. flagelliforme in the high salt stress condition. Finally, the metabolic regulation mechanisms of Nostoc flagelliforme in response to high salt stress would be uncover based on the metabolic reprogramming, and the key metabolites associated with the high salt stress could be discover. This study could provide reference for the study of the salt tolerance mechanism of terrestrial cyanobacteria. Further more, the identification of key metaoblites may serve as a novel target for salt resistance characteristics manipulation of N. flagelliforme, which could provide the theoretical foundations for the resource protection and directional improvement of strains.

发菜（Nostoc flagelliforme）是一种可食性陆生固氮蓝藻，主要分布于沙漠和贫瘠土壤中，具有重要的资源价值和生态价值，逆境胁迫是其生长过程中不可避免的环境因素。然而，目前关于发菜抗逆的分子机制尚不清楚，尤其是基于小分子化合物的代谢调控机理方面的研究较为缺乏。在前期工作中，申请人已初步建立了基于GC SIM/MS和UPLC MRM/MS的发菜“拟”靶向代谢组学分析方法。本研究拟在此基础上，进一步结合同位素示踪技术，发展稳定同位素标记的“拟”靶向代谢组学分析策略，监测高盐胁迫下发菜细胞代谢物的动态变化过程，从代谢重编程的角度探讨发菜响应高盐胁迫的代谢调控机制，并筛选关键调控分子，从代谢物与代谢组及生物大分子互作关系的视角对其生物功能进行初步阐明。本研究可为陆生蓝藻抗逆机理的研究提供参考；筛选所得的关键调控分子有望作为发菜耐盐性能调控的新靶标，为资源保护及菌株改良提供理论依据。

发菜是一种名贵可食的陆生蓝藻，富含藻胶、蛋白质钙、铁等营养成分，具有较高的营养价值和药用价值。发菜主要分布于世界各地的沙漠和贫瘠土壤中，逆境胁迫是其生长过程中不可避免的重要因素。为了保护和开发利用发菜这一特色生物资源，开展发菜逆境胁迫机制的研究显得尤为重要。然而目前对发菜抗逆机制的研究主要局限于生理生化方面及某一代谢途径中局部基因和蛋白等方面，基于小分子化合物的代谢调控机理方面的研究较为缺乏。在青年基金的资助下，研究团队以人工液体培养发菜为研究对象，考察不同盐浓度及胁迫时长对发菜细胞生理生化水平的影响及代谢特征的影响。盐胁迫时液体培养发菜细胞链弯曲，异形细胞增多，营养细胞自溶并逐渐消亡。盐胁迫后，叶绿素a和藻蓝素的含量降低，胞外多糖、质膜透性和丙二醛含量增加，超氧化物歧化酶活性在低盐胁迫时升高，高盐胁迫时降低。这表明盐胁迫抑制了液体培养发菜的光合作用，增加了发菜细胞膜的通透性，使其发生过氧化反应，导致细胞膜受损。机制研究发现发菜细胞在盐胁迫下大多数氨基酸含量上调，脂肪酸含量下调。脂质代谢分析发现，盐胁迫对人工液体培养发菜的鞘脂、磷脂和糖脂代谢途径产生了显著影响，其中盐胁迫后，发菜细胞中神经酰胺、单半乳糖甘油二酯和磷脂酰甘油含量高于对照组，而磷脂酰胆碱、磷脂酰乙醇胺及单半乳糖基单酰基甘油酯含量低于对照组。本项目从代谢重编程的角度探讨发菜响应高盐胁迫的代谢调控机制，并筛了选关键调控分子，阐明其生物功能。本研究可为陆生蓝藻抗逆机理的研究提供参考；筛选所得的关键调控分子有望作为发菜耐盐性能调控的新靶标，为资源保护及菌株改良提供理论依据。