超低温保存是植物种质资源离体保存的重要途径，保存过程中低温、脱水等复合胁迫引起胞内ROS失衡、质膜损伤、蛋白变性、离子毒害等严重影响细胞活性。脱水素与植物多种胁迫响应密切相关，具有保护核酸、蛋白与膜结构的功能。申请人前期在百子莲胚性细胞超低温逆境应答研究中筛选到2种具有重要保护作用的脱水素蛋白(Y2SK2与SK3型)，其结构与应答模式均具差异，但作用机制尚不明确。本项目拟通过启动子克隆、调控元件分析探明2个蛋白的逆境应答机制；依据亚细胞定位与免疫电镜技术确定蛋白的胞内作用靶点；应用体内、外蛋白功能验证，以及过表达与RNAi技术，结合蛋白质组、生物物理、分子结构与热力学分析，揭示2个蛋白对质膜结构、ROS代谢、抗氧化、离子亲和、关键酶活、冰晶抑制、结构相变的调控作用；解析脱水素在复合逆境下的结构与功能特性，阐明其对细胞的保护机制，丰富植物种质资源保存理论体系，为改善植物离体保存效率提供新思路

Cryopreservation is an important technique for in vitro conservation of plant germplasm resources, which is a combination of low temperature biology and micro propagation technology. Low temperature and dehydration stress leading reactive oxygen species (ROS) metabolism imbalance, plasma membrane damage, protein denaturation, ion toxicity, and these events affect cell viability seriously during cryopreservation. In our previous study, we using transcriptomic and proteomics technology uncover the stress response mechanism of Agapanthus praecox embryogenic callus cryopreservation, and screened 2 important dehydrin proteins, Y2SK2- and SK3-type, which play a key role in abiotic stress response and protecting the cell activity during cryopreservation. Dehydrin (DHN) can protect nucleic acids, protein and membrane structure, which is closely related to the stress tolerance in plants. This project intends to explore the mechanism of DHN expression regulation by the promoter cloning and cis-acting element analysis; to determine the subcellular localization of the protein by immuno electron microscopy and protein transient expression skills; furthermore, application of in vivo, in vitro protein function validation, gene over-expression, RNAi and proteomics technology, combined with biological physics, molecular structure and thermodynamics analyses reveal the protein protective effect on membrane system, ROS metabolism, antioxidant system, ion affinity, enzyme activity, ice-crystal inhibition, and phase transition temperature. Elucidate the structural and functional differences and cell protective mechanism of Y2SK2- and SK3-DHN under complex stress. This study will provide a new insight for improving plant stress tolerance, and supplement theoretical foundation for plant germplasm resources preservation and utilization.