超低温保存过程中ROS介导的细胞凋亡对百子莲胚性愈伤组织冻后成活率的影响机制

超低温保存是植物离体资源中长期保存的最佳途径。申请人前期研究表明百子莲胚性愈伤组织（Embryogenic callus，EC）超低温保存过程中发生明显的细胞凋亡，而活性氧簇（ROS）介导的氧化胁迫与细胞凋亡（Apoptsis）是影响植物冻存细胞成活率的主要因素，揭示相关的分子作用机制是低温生物学领域的一个重要科学问题。本课题旨在通过ROS与细胞凋亡的原位检测和生理生化指标测定明确ROS代谢水平、细胞凋亡程度与细胞成活率的相关性，筛选具有主要调控作用的ROS信号分子、抗氧化酶与内源抗氧化剂的种类。应用RNA-seq与iTRAQ技术在转录组学与蛋白组学层面构建ROS诱导细胞凋亡的信号调控网络，并明确参与细胞凋亡的蛋白酶类型。利用外源调控物质从信号转导途径上游调控ROS水平，下游缓解细胞凋亡的发生程度，进一步丰富植物超低温保存理论，为最终提高冻后细胞成活率的体系优化提供参考。

Cryopreservation is the best way to plant vitro resources long-term preservation. How to improve plant cryopreservation survival rate is an important scientific issue in the cryobiology field. Some researches indicate that reactive oxygen species (ROS)-induced oxidative stress and apoptosis may become the main factors in immediate and delayed onset cryoinjury. Furthermore, our preliminary studies showed that the embryogenic callus (EC) occurs significant apoptosis during cryopreservation in Agapanthus praecox. This project aims to identify the dynamic changes of ROS and apoptosis during cryopreservation using cell in situ skill and physiological indicators determination, to reveal the the correlation between ROS, apoptosis and cell survival rate, and screening the major role of ROS molecules, antioxidant enzymes and endogenous antioxidants in Agapanthus cryopreservation. Moreover, RNA-sequencing and iTRAQ (isobaric Tags for relative and absolute quantitation) technologies were designed to screen differentially expressed genes and proteins of Agapanthus EC during cryoperservation, and establish ROS-induced apoptosis signaling regulatory network at transcriptomic and proteomic levels. Eventually application valid exogenous antioxidants and protease inhibitors regulate ROS levels at upstream and mitigate apoptosis at downstream of ROS-induced apoptosis pathway to improve cell survival rate of cryopreservation. This project has important scientific significance to promote theory innovation and technological advances in cryopreservation field.