申请人前期将单壁碳纳米管(CNT)添加于玻璃化溶液可明显提高百子莲胚性细胞(EC)超低温保存冻后活性，发现CNT进入细胞主要贴膜富集于线粒体，对呼吸代谢具有积极影响，线粒体作为EC中最为重要的细胞器，但CNT对其功能的调控机制尚不清楚。本项目拟基于CNT超低温保存逆境保护生物学效应模型，在细胞结构学层面分析CNT对线粒体形态与膜功能的影响，评价逆境下CNT与线粒体结构的互作效应；在生理、基因与蛋白层面研究CNT对呼吸代谢途径的影响，并采用呼吸酶抑制剂确定CNT在呼吸链的作用位点；利用线粒体体外系统进一步功能验证，分析CNT对线粒体响应关键胁迫因子的调控作用；建立CNT与其调控的关键线粒体相关生物大分子互作的研究体系，使用分子动力学模拟CNT与其作用蛋白之间的结构动力基础。利用多学科研究手段揭示CNT在改善超低温保存效率过程中的线粒体生物学效应，为纳米材料在超低温保存领域的应用提供理论依据。

Cryopreservation is the best way for long-term preservation of plant germplasm resources, and how to improve plant cryopreservation survival rate is an important scientific issue in the cryobiology field. Our preliminary studies found that adding single-wall carbon nanotubes (SWCNT) to plant vitrification solution (PVS) significantly improved the survival rate in Agapanthus praecox embryogenic callus (EC) cryopreservation, and SWCNTs were mainly concentrated in mitochondria and cell membrane. The SWCNT had a positive role in the regulation of cell respiration under stress. As the most important organelle in EC, the mechanism of interaction between SWCNT and mitochondria function was unknown. This project analyzes the effects of SWCNT on mitochondrial morphology configuration and membrane function at the cytological level. In addition, the effects of SWCNT on the respiratory metabolism pathway during cryopreservation are studied by physiological analysis, qRT-PCR and MRM, and the respiratory enzyme inhibitors are used to determine the action site of SWCNT in the respiratory chain. Further functional verification of mitochondrial in vitro system is used to analyze the regulatory role of CNT on the key stress factors of mitochondrial response. Furthermore, this project establishes the experimental study system to analyze the interaction between SWCNT and its key biological macromolecules including structural characteristics, binding mode, active site and so on. Then the project uses an all-atom model to simulate the interaction between the SWCNT and key protein. This project aims to reveal the biological effects of SWCNT on mitochondrion and the mechanism of SWCNT improving cell survival rate in cryopreservation of Agapanthus praecox EC based on interdisciplinary analyses, and has important scientific significance to promote theory innovation and technological advances in cryopreservation, and lays the foundation for the research of the interdisciplinary field of nanobiology.