玻璃化法超低温保存是百子莲胚性愈伤组织（EC）中长期保存的最佳途径。申请人前期研究发现，百子莲LR型丝氨酸蛋白酶抑制剂ApSerpin1在超低温保存中表达差异显著，其原核表达的蛋白是百子莲EC超低温保存的有效优化物质，使冻存率提高了近30%。本项目拟在已获得的ApSerpin1重组蛋白的基础上，通过生理生化检测揭示外源ApSerpin1对百子莲EC超低温保存中细胞程序性死亡和氧化响应机制的调控作用；应用免疫胶体金定位技术明确ApSerpin1的作用位点；构建ApSerpin1过表达和RNA干扰的百子莲转基因EC验证其分子功能；利用CoIP、pull-down、BiFC等技术通过基于互作关系阐明ApSerpin1调控百子莲EC超低温保存的分子机制，以期为ApSerpin1作为冷冻保护剂添加剂的开发奠定理论基础，最终实现生物样本的离体保存。

Vitrification-based cryopreservation is the most effective technique for cryopreservation of Agapanthus praecox embryogenic callus (EC). In the previous research, applicant identified a gene from A. preacox named ApSerpin1, coding a LR-type serine protease inhibitor, differentially expressed in cryopreservation process. Application of its recombinant protein from E.coli significantly improved the survival rate of A. praecox EC for nearly 30%. In this study, we will further analysis the regulation mechanism of ApSerpin1 on programmed cell death process and oxidative response of A. praecox EC cryopreservation. For investigate its mode of action, immuno-gold localization was performed to identify the subcellular localization of ApSerpin1. Transgenic EC of ApSerpin1 overexpression and RNA interference were obtained to demonstrate its molecular function. Furthermore, the proteins-interaction between ApSerpin1 and different targeted proteins was to elucidate the functional mechanism in cryopreservation using CoIP, pull-down and BiFC techniques. This work aimed to lay the foundation of the theoretical basis of ApSerpin1 as a cryoprotectant additive and provide a reference solution for in vitro preservation of other biological samples.