| Most seafood had been transported in the oxygenated ice water in the pastalthough this method had the high cost, quality damaged and high security risks.Currently anhydrous transportation at low temperature is more widely applied to thetransport of marine fish owing to its’ low costs and keeping animal’s alive.Turbot iswidely farmed in northern China.By studying the change of serum physiological,biochemical parameters and gene expression mechanism during its cooling process,,we can improve the method of transport, extend transport time which will bebeneficial to the application and economic. In view of this, we studied the continuouscooling effect on the turbot serum biochemical and molecular parameters to explorethe cold stress tolerance of turbot indicate the transportion.1. Effect of continuous cooling on serum physiological, biochemicalindex of adult Scophthalmus maximus LSerum physiological and biochemical indicators tested by biochemical methodscan reflect metabolism and stress response of turbot during coolingprocess.Then,sensitive indicators were chosen from those. The main results are asfollows: serum glucose and cortisol level elevated significantly, indicating the bodyproduced a stress response to cool, serum alanine aminotransferase, lactatedehydrogenase activity change indicating the function of liver and heart was affected,lactate in muscle significantly elevated reflecting that aerobic respiration was affected,and the remaining tissue was hardly affected.2. Effect of continuous cooling on the expression of HSP70andHSP90of adult Scophthalmus maximus LBy real-time PCR techniques, the the expression levels of related genes were detected to explore the correlation between temperature decreasing and related genes.Thirdly, Wap65gene was cloning and its’ expression levels were detected in thecooling process which enriched molecular biological indicators reflecting to thetemperature change. The main results are as follows: the HSP70expression is affectedby the tissue and temperature. Heart, spleen, brain and head kidney to have stressself-regulating ability to low temperature. Liver and kidneys have a certain stressresponse only on beginning. Change of HSP70expression in the intestinal tissue is noregular,so these tissues are not suitable to be the indicator of temperature stressresponse. In the gills and stomach, the expressions of HSP70are sensitive to thetemperature decreasing, so they can be the indicator. HSP90has stronger tolerance tolower temperature than the HSP70, at1℃HSP90in most organizations be able toresume near to the initial level. However, the expression of HSP70in gill increasedsignificantly with temperature decreasing, therefore HSP70is more suitable to be amolecular indicator.3. Effect of continuous cooling on the expression of Wap65of adultScophthalmus maximus LFragment wap65was cloned in liver tissue. After blasting with other species,fragment sequences have been submitted to the NCBI database, GenBank accessionKF879928(unpublicized) is given. The expression of wap65is studied by real-timePCR showing that the continuous cooling had an effect on all tissues (brain, stomach,liver, head kidney, kidney, spleen, muscle, intestine at a specific temperature, gills,heart). The expression of wap65in them increased significantly during cooling.Stomach, liver, muscle and intestinal tissues hardly changed in contrast that headkidney, kidney and spleen tissue changed significantly, indicating that the Wap65inthose tissues are sensitive to continuous cooling.Sensitive indicators of continuous cooling of turbot are selected. Lactate in musclesignificantly increased with temperature decreasing. Serum glucose and cortisol alsosignificantly increased, so they all can also be used as the sensitive index totemperature decreasing of turbot. HSP90had strong ability to adapt to temperaturechanges, so it was not suitable to be the index. HSP70is sensitive to the temperature lowering, so it can be used as the molecular index of turbot. Gene fragments Wap65isthe first cloned in turbot. In head kidney, kidney and spleen tissues, Wap65gene issensitive to temperature change however it is not sensitive in other organizations. |
