The Study Of Anti-adversity Mechanism In Hemolymph Of Sinonovacula Censtricta Lamark With Proteomic Approaches

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.