无水保活流通过程中南美白对虾冰温损伤的靶点识别及应答机制

Structural damage and acute mortality of L. vannamei induced by ice-temperature stress during anhydrous living- transportation is the bottleneck for living circulation and it is vital important for reducing the ice-temperature injury to elucidate the biomarker and responding mechanism. As the special tissue responding to environmental stress in L. vannamei, hepatopancreas is the most sensitive organ to injure and the protein expression profiling and metabolic regulation network are closely associated with the effect of ice-temperature stress. In this project, two dimensional electrophoresis approach is used to find the differentially expressed proteins and explicit the change law in expressional patterns of these proteins. With Mass spectra analysis and Western blotting technique, the differentially expressed proteins are identified and verified at molecular level. By correlation analysis of profile changes in protein expression with injuring effect, the target proteins are recognized. Then changes in functional patterns are further elucidated and the protein molecular mechanism of L.vannamei respond to cold stress damage is revealed by bioinformatics analysis. Furthermore, the metabolites in hepatopancreas of L. vannamei during anhydrous living-transportation are investigated with metabonomics strategy and the changes in metabolite fingerprint spectrum are analyzed with pattern recognition and nonparameter text to identify the biomarker respond to ice-temperature stress. The metabolic pathway of typical biomarkers and their dynamic interaction network are established according to bioinformatics analysis. The target proteins and biomarker metabolites are integrally investigated with bioinformatics analysis to explore the metabolic pathway and interactive regulation network. By quantitative analysis of relationship between injuring effect, target proteins and metabolites, the molecular injuring biomarker and responding mechanism are revealed and the theoretical basis is established for early warning and control against cold damage of L. vannamei during anhydrous living-transportation.

冰温胁迫诱导的应激性损伤和猝死是制约南美白对虾无水保活流通的瓶颈，明确冰温损伤分子靶点揭示应答机制是冰温损伤控制的关键。而肝胰腺作为对虾应答环境胁迫的效应标记器官，其功能蛋白分子的表达、代谢产物调控与冰温损伤效应密切相关。为此，本项目首先应用双向电泳技术寻找冰温胁迫下肝胰腺差异表达蛋白，在质谱鉴定的基础上，运用蛋白质印迹技术对关键差异蛋白进行验证，结合存活质量变化明确冰温损伤靶点蛋白。然后运用代谢组学技术分析肝胰腺代谢轮廓指纹图谱变化趋势，采用模式识别与非参数检验对数据进行主成分分析和功能聚类，结合冰温损伤效应，明确关键冰温应答代谢标志物。进而将冰温损伤靶点蛋白及代谢标记物进行生物信息学分析，明确代谢途径及其互作网络变化规律，分析损伤效应与靶点蛋白及代谢响应间定量关系，阐明冰温损伤分子靶点及应答机制，为南美白对虾无水保活流通过程中冰温损伤早期预警及控制奠定理论基础。

模拟南美白对虾无水保活流通实际，系统考察了降温及生态冰温条件对存活及肌肉品质的影响，并从生理生化、氧化应激与免疫响应、肝胰腺细胞结构变化、蛋白质组和代谢组角度解析存活差异的整体机制。结果表明：（1）调整降温及生态冰温条件可显著延长无水存活时间；（2）肌肉品质随双重胁迫时间延长而有不同程度下降；（3）血清皮质醇、葡萄糖、乳酸、血蓝蛋白、尿素氮和肌酐含量均增加，总蛋白先升后降，总胆固醇和甘油三酯呈时间依赖性下降，表明双重胁迫诱导的生理应激可造成对虾物质代谢紊乱；（4）双重胁迫进程3h前糖酵解反应加快，之后糖异生反应加强，至9h代谢紊乱，提示胁迫进程中机体发生了一系列代偿性调节；（5）ROS和MDA含量显著高于对照组，且在第6h达到最高，SOD、CAT和GSH-Px活力均随胁迫时间延长而增加，非特异性免疫酶PO活力显著增加而POD整体下降，ACP和AKP逐渐上升，表明机体通过激活抗氧化酶和非特异性免疫酶应答环境胁迫；（6）组织病理显示双重胁迫9h时肝小管有轻微解体，内壁细胞部分脱落，超微结构进一步表明了细胞器损伤，提示肝胰腺组织不可逆损伤可引起对虾死亡；（7）蛋白质组分析共鉴定到968个蛋白，9个蛋白上调和24个蛋白下调，溶酶体、辅因子生物合成、抗坏血酸盐代谢和细胞凋亡途径是机体响应双重胁迫的关键途径。（8）代谢组学分析发现双重胁迫环境下代谢标记物和代谢途径与正常组显著不同，差异主要在神经配体-受体互作通路、雌激素信号通路、促性腺激素释放激素分泌途径、氨基丁酸能突触途径、内源性大麻素反馈途径、cAMP信号途径。本研究整体识别了南美白对虾无水保活流通过程中的冰温损伤靶点及机体响应机制，为后续保活流通存活质量监控和参数完善提供了理论基础。