【作者】 张鹭；

【导师】 陈寅山；

【作者基本信息】 福建师范大学 ， 动物学， 2003， 硕士

【摘要】 以闽、浙一带重要的海产养殖贝类缢蛏（Sinonovacula constricta Lamarck）为研究对象，应用蛋白质组学方法探求其在高温、低盐、受寄生等外界环境压力下血淋巴内40KDa以下小分子蛋白质组的变化，小分子碱性蛋白的变化，寻找缢蛏血淋巴的抗性机制，明确血淋巴在机体防御体系中的作用。同时也对双向聚丙烯酰胺凝胶电泳过程中肌肉样品的制备方法进行优化。 在环境压力下，缢蛏血淋巴内有多种蛋白发生变化，包括生长因子，DNA结合蛋白，锌指蛋白，金属硫蛋白，磷脂酶A₂等，涉及多种与炎症反应，基质转运，神经营养，基因表达调控，蛋白质合成等有关的功能。说明血淋巴在缢蛏的防御体系中具有关键性作用。通过比较研究，也优化了肌肉样品双向电泳样品制备方法及电泳系统。本研究证明了蛋白质组理论及技术在分析生态学问题时的适用性及灵活性。更多还原

【Abstract】 Background Sinonovacula constricta (Lamarck), a kind of mollusks, is important in marine aquaculture due to its high market demand. They have eurythermal (0-3 9 癈) and euryhaline (4-40%o) potteries. So they can widely distribute throughout pebble beaches among China and be fed easily. We choose S. constricta as our experiment sample.To data, there have been many reports illustrating the differential protein expression in response to salinity, temperature and antoeciousness stress. Unfortunately, Seldom work on differential protein expression of bivalve mollusk hemolymph after environment disturbances was reported, and there is no report on parasitism-specific protein from S. constricta hemolymph. Most of those studies are based on radiolabelling and chromatography techniques and proteins separation in SDS-PAGE. Recent improvements in proteomic techniques have facilitated the analysis of molecular mechanism on protein level. Proteomics research has become one of the landmarks for era of post genome and it’s useful in many areas of biology medicine and pharmaceutics. It has been also used to analysis ecological issues recently.Objective The purposes of this study are (i) to detect whole and basic protein expression profiles in hemolymph after short-term treatment of high temperature and low salinity respectively, and to detect the parasitism-specific proteins under long-term parasitizing (ii) to identify differential proteins for these three ecological factors, then inference mechanisms about hemolymph resistance through a series of experiments, (iii) to take further steps to demonstrate the feasibility of proteomics procedure on ecological issues.Methods and Steps We use two-dimensional electrophoresis and image analysis software to detect differential protein expressing under various stresses, then cut theinterested protein spot and digest it with trypsin. We can get peptide mass map with MALDI-TOF-MS. A novel protein or polypeptide thus is identified by comparing with Corresponding map of known sequences in database.Our study included two parts: (i)Establishing of method systems of 2- DE. We choose S. constricta adductor muscle as object, to preliminarily explore sample preparation and electrophoresis systems, (ii) detect various differential protein under ambient disturbances. We specified basic proteins to avoid their being shielded by high abundant proteins . In all our studies.we pay more attentions on low mass proteins under 40Kd because they always are highly bioactive.Conclusions Invertebrate, lack an adaptive immune system, hemolymph can act as defense barrier and maintain relatively constant conditions through homeostasis. Our results proved former conclusions partly, and they provided detail evidences that S. constricta hemolymph involve plenty of proteins with multiple functions such as nutrition, gene expression, regulatory protein synthesis, substances transport, antitoxin, anti-interference. According these proteins, It suggested that S. constricta can choose proteins as effective adaption to ambient disturbance and it can resist the ecological interfere at tissue, cell, protein and even gene levels. It’s obvious that hemolymph is critical in S. constricta defense mechanism.Among three ecological factors, parasitism can influence S. constricta hemolymph most seriously, and it seems that S. constricta is more sensitive to low salinity than high temperature.Our results also illustrated the usefulness and superiority of the proteomics technologies to analy ecological molecular mechanism.更多还原