脱水素蛋白在超低温复合逆境中对百子莲胚性细胞的保护机制研究

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.

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

本项目研究百子莲两种脱水素对非生物逆境与激素信号的应答模式与调控机制，通过转基因与原核表达技术揭示蛋白构象变化的结构基础和生物大分子的互作机制，阐明在细胞体内与体外的生理保护功能。探索植物脱水素蛋白在改善植物抗逆性、离体细胞保存，细胞抗氧化、抗衰老等领域应用的可行性。.两个脱水素表达具有组织特异性，均能积极响应干旱、高渗、盐、低温、ABA信号，ApY2SK2逆境应答模式为：高渗>低温>ABA>盐>干旱>高温；ApSK3为：盐、ABA>渗透、低温、干旱>高温。ApY2SK2的响应敏感性强于ApSK3。两个蛋白定位于细胞质和细胞膜，ApY2SK2具有核定位信号。.ApY2SK2与ApSK3过表达植株抗逆性明显改善。不同胁迫下，膜脂过氧化程度和ROS水平降低，抗氧化活性、脯氨酸和光合能力显著提升。转基因拟南芥超低温复合逆境下的存活率较野生型冻存提升一倍以上。ApY2SK2与ApSK3转化百子莲胚性愈伤组织，不同基因型胚性细胞超低温冻后活力具显著差异。过表达细胞系死亡率降低，H2O2含量显著降低, 氧化应激水平增强；RNA干扰细胞死亡率上升，MDA与OH·水平显著升高，氧化胁迫严重。不同细胞系进行蛋白质组学与酶活分析表明脱水素可有效保复合逆境系下胞内抗氧化酶等蛋白活性。.体外实验证明ApY2SK2与ApSK3在冻融和脱水状态下可有效保护其他蛋白活性；两种蛋白均能够结合金属离子并抑制Fenton反应中羟自由基产生，离子结合能力为Co2+ >Ni2+ >Cu2+ >Fe3+。脱水素蛋白通过将α-螺旋转变为β-转角增强与质膜结合作用，缓解脂膜破裂与融合积聚现象，提高质膜系统稳定性。应用脱水素蛋白体外添加冷冻保护液优化植物超低温保存体系，使幼苗存活率由20%提升至50%左右。综合本项目研究内容， ApY2SK2与ApSK3在体内与体外均能有效保护细胞抵御逆境胁迫伤害。