两种蛋白酶在百子莲胚性愈伤组织超低温保存诱发的细胞程序性死亡中的作用机制

Our early studies suggested that the cryopreservation-induced PCD in Agapanthus praecox embryogenic callus (EC) mainly occurred during dehydration to 24-h recovery phases. The expression of proteinases, which are the initiator and executer of PCD also significantly changed during this period. While the mechanism of two proteinases reducing the cell viability has rarely been reported. This study intends to established the relationship among the activity of proteinases, PCD and cell viability or death ratio during rewarming to 0~72h recovery phases through the measurement and analysis of the TTC and Evans Blue staining, mitochondrial membrane potential and activities of caspase-like, along with the detection of FDA fluorescence, TUNEL and Hoechst-PI fluorescence and DNA ladder. Based on an established post-thaw model of Arabidopsis seedlings, the seedlings at different recovery stages are screened to identifity the key proteinase that are involved in PCD. The function of key proteinase then is verified via analysis of amino acid sequences and domains, in vitro enzymolysis experiments and addition of specific inhibitors. The interaction mechanisms of interaction of up- and downstream proteins in cryopreservation- induced PCD pathway is revealed using promoter cloning, yeast twohybridization, CRISPR/Cas9 and iTRAQ technologies. The prospective results would enrich theoretical system of key proteinase in plant cryopreservation, and provide new insight and scientific basis for improving efficacy of exogenous protective substances added to the cryopreservation culture conditions.

百子莲（Agapanthus praecox）胚性愈伤组织（EC）超低温保存诱发的细胞程序性死亡（PCD）主要发生在脱水至冻后恢复培养24h，且有延迟性特征，导致细胞活力下降，但相关机制鲜见报道。本项目前期工作发现丝氨酸和半胱氨酸蛋白酶在超低温保存中表达量显著提高，为此，拟以冻后恢复培养0~72h的百子莲EC为样品，检测TTC/Evans Blue值、线粒体膜电位和类Caspase酶活性，辅以FDA、TUNEL和Hoechst-PI检测、DNA ladder电泳，构建蛋白酶活性、PCD与细胞活力间的关系。通过蛋白酶结构域分析、体外酶解、外源抑制剂和拟南芥超低温保存成活率筛选体系分析蛋白酶的功能；染色体步移技术克隆及分析蛋白酶启动子，酵母双杂及CRISPR/Cas9基因敲除技术揭示蛋白酶与上游转录因子和下游蛋白的互作机制，从而为开发新的蛋白酶抑制剂提高植物资源保存成活率提供理论基础。

百子莲胚性愈伤组织（EC）超低温保存过程中会诱导细胞程序性死亡（PCD）发生，包括冻存处理导致的PCD和恢复培养0~36h内发生的延迟性PCD。恢复培养6h时，发生最为严重的细胞凋亡和细胞坏死，至18 h逐渐减弱，在24~36 h又进一步加剧，此时以细胞坏死为主。通过类Caspase-1、-3、-6和-8活性、MDA和ROS水平检测及指标相关性分析，表明H2O2是延迟性PCD的主要诱导因子之一，类caspase-3与细胞凋亡具有显著正相关关系。ApCathB和ApSBTs均为影响百子莲EC冻存率的关键基因。ApSBT具有2个可变剪切体，其中ApSBT.1为全长基因编码的蛋白，ApSBT.2为第三内含子保留而引入提前终止密码子（PTC）编码的截短蛋白。两个剪切体的编码蛋白亚细胞定位不同，ApSBT.1加剧并且提前了超低温保存过程中PCD发生的进程，而ApSBT.2对恢复培养阶段减弱PCD进程明显。ApSBTs启动子含有ARE、MBS、MYB、G-box等顺式作用元件，表明ApSBT的表达与胁迫响应和激素信号相关，还可能受到MYB转录因子的调控。过表达ApCathB的拟南芥和百子莲EC超低温保存过程中PCD加剧，而干扰表达ApCathB减弱了恢复培养阶段的PCD进程。ApCathB主要通过提高百子莲EC中组织蛋白酶B活性，激活下游类caspase-1和类caspase-3后促进PCD的进程。加剧发生的PCD造成体内ROS累积，高水平的ROS则进一步降低了细胞冻存率。利用RNA-seq分析构建了ApCathB参与的转录调控网络图，ApCathB通过调控细胞壁和蛋白降解、水解酶等蛋白加剧细胞内蛋白降解作用，引发内质网胁迫产生并诱发PCD加剧发生。因此，通过添加小分子抑制剂E64及内源蛋白酶抑制剂ApCystatin，能够有效提高植物组织的冻存率，有望开发为一类新型的冷冻保护剂。