丝氨酸蛋白酶介导的酚氧化酶原激活诱发冷藏对虾黑变规律与机制

Activation of prophenoloxidase activating system (ProPO-AS), an important innate immune system in crustaceans such as shrimp, can lead to melanization and thus is mostly responsible for the shrimp quality degradation during cold storage. In the present research, the serine protease (SP), the critical constituent in ProPO-AS, is firstly investigated, and with respect to the tissue specificity of SP spatial distribution, the changes in activity and location of SP during cold storage is monitored by immunity enzyme cyto-chemical technique and electronic microscope. To reveal the activation pattern of ProPO-AS mediated by SP during cold storage, the development of and relationship between the Ca2+ concentration, SP activity and the ProPO activation degree are subsequently determined by techniques of laser scanning confocal microscope and enzymatic activity determination. Meanwhile, SP and ProPO, the critical constituents in ProPO-AS, are purified from fresh shrimp plasm by affinity chromatography and the in vitro model is constructed. More exactly, the conformation change during cold storage is further characterized by UV, fluorescence and circle dichromatism spectrum techniques and electrophoresis technique. And the activation mechanism of ProPO is explored by combining the conformation changes with the activity status of ProPO in shrimp during cold storage. Results of this study provide not only a theory basis for target inhibition of shrimp melanization during cold storage, but also deepen the insight into ProPO-AS in shrimp.

酚氧化酶原激活系统(ProPO-AS)是对虾等甲壳类动物固有免疫系统的重要组分，但ProPO-AS激活会引起黑变，是对虾冷藏期间品质劣变的主要原因。本研究以ProPO-AS关键性组分丝氨酸蛋白酶(SP)为突破口，鉴于SP空间分布的组织特异性，采用细胞免疫化学和透射电镜技术跟踪SP在冷藏期间的组织迁移变化。继而采用激光共聚焦偶联酶活检测跟踪冷藏期间组织内Ca2+浓度、SP活性和ProPO激活程度的动态变化，分析变量之间的相互关系；同时通过免疫亲和技术从对虾中分离SP和ProPO，构建包含主要组分的体外激活模型，揭示冷藏期间SP介导的ProPO激活规律。进而运用紫外、荧光和圆二色等光谱技术和电泳技术解析对虾冷藏期间ProPO的分子构象和聚集行为，结合系统活性状态，探讨SP介导的ProPO激活机制。本研究不仅可为对虾冷藏期间的黑变靶向抑制提供理论依据，而且将深化对ProPO-AS的理论认识。

酚氧化酶原激活系统(ProPO-AS)是对虾等甲壳类动物固有免疫系统的重要组分，但ProPO-AS 激活会引起黑变，是对虾冷藏期间品质劣变的主要原因。本研究首先通过感官分析和色差测定明确了对虾冷藏期间感官品质劣变的关键时间点和控制部位，分析了南美白对虾冷藏期间的黑变速率与内源多酚氧化酶及丝氨酸蛋白酶酶原激活水平间的相关性，并运用低场核磁共振、透射电镜和组织化学技术进一步探讨其生化机制。结果表明，南美白对虾4℃冷藏期间的黑变具有部位和时间特异性，头部为黑变最敏感部位，其黑变速率与内源多酚氧化酶和丝氨酸蛋白酶活力水平密切相关，冷藏第四天时样品失去商品价值，此时酚氧化酶活力达到实验期间的最高水平，丝氨酸蛋白酶激活水平与酚氧化酶变化趋势具有一致性，低场核磁共振结果表明随着冷藏时间的延长，头部结合态水分驰豫时间T21减少而游离态水T22增加，组织化学和透射电镜显示肝胰腺组织和细胞结构在冷藏期间均有显著破损，免疫组织化学结果进一步表明由于胞浆内质网解体而引起Ca2+呈弥散状释放，同时丝氨酸蛋白酶呈现出与水分和Ca2+迁移相一致的组织迁移变化，电泳和光谱学分析表明在对虾冷藏过程中肝胰腺组织酚氧化酶的电泳行为和聚集状态发生了显著变化，其特征与酶活变化和黑变进程有良好的对应关系。综合感官评价、酶活监测、组织化学、电泳和光谱学分析结果证明丝氨酸蛋白酶激活系统介导了对虾冷藏过程中酚氧化酶酶原的激活，从而引发和加速了黑变进程。以此为基础，本项目进一步就典型冷链温度下微生物菌相变化及其肠道微生物宏基因组提取方法、品质劣变动力学模型及其绿色控制进行了拓展研究，明确了CTAB法为肠道微生物宏基因组优选提取方法，揭示了典型冷链温度下的优势微生物及其动态变化规律，构建了以整体稳定性GSI为指标的品质劣变动力学模型、证明了虫草多糖和饥饿胁迫处理可有效减缓黑变进程。本项目不仅整体上明确了冷藏对虾黑变的动力学规律和生化机制，而且为品质劣变的绿色控制提供了技术途径。