Comparison Research Of Different Fish Based On Two-Dimensional Gel Electrophoresis Technology

In recent years, two-dimensional gel electrophoresis technology has been gradually applied to researches of fish production safety, especially in the identification of fish species, physical and chemical evaluation of fresh fish, meat quality changes during fish storage and processing, difference test of batch quality, standardization of fish product. In view of this, this paper uses Pseudosciaena crocea as the typical study object, optimizing procedures through Pseudosciaena crocea muscle tissue sample handling, hydration loading mode, IEF procedure and staining methods, based on introduction of two-dimensional gel electrophoresis technique. After exploring various conditions, finally establish two-dimensional gel electrophoresis method for fish muscle protein. Based on it, this paper uses Pseudosciaena crocea, Laieolabrax japonicas, Ctenopharyngodon idell and Crucian carp as the research objects for comparing different fish quality (differential proteins, biochemical composition, textural properties, microstructure and so on), combining with two-dimensional gel electrophoresis technology. The main results are as follows:1. Objective of the research is aimed to construct treatment method of muscle proteins of Pseudosciaena crocea for two-dimensional electrophoresis (2-DE). The protein samples of Pseudosciaena crocea muscle tissue are extracted. Then the extraction is used for two-dimensional electrophoresis (2-DE) for original trial. We also compare and optimize different vital factors and procedures. The final optimized extraction liquid content is8mol/dm3Urea,2mol/dm3Thioruea,4%CHAPS,0.2%Bio-Lyte ampholyte. The p H of17cm IPG gel is5-8, loading volume is160μg. After series of trials, we finally get satisfactory2-DE pattern. The loading mode is active loading mode, the IEF program is slow raising voltage:firstly,12hours of50V active loading mode, then500V1.5h,1000V1h,4000V1.5h,4000V0.5h,8000V h,500V10h, loading and IEF program is processing at20℃After a series of trials, we finally get satisfactory2-DE patterns with high quality and repetition. With optimization of these conditions, the technique of2-DE has been successfully established.2. Compare different proteins of Pseudosciaena crocea, Lateolabrax japonicas, Ctenopharyngodon idell and Crucian carp, combining with established2-DE method. After the PDQuest softwate analysis, the repeatability (n×3) of triplicate gels for each sample was more than70%(p<0.05). More than800spots are detected on each silver stained gel. The result indicated that68spots showed significant difference among fish by using statistical and quantity analysis. The differently expressed proteins of Pseudosciaena crocea and Lateolabrax japonicas were distributed in the region of MW30-52kDa, pH7-3and MW40-55kDa and pH5-3. The differently expressed proteins of Ctenopharyngodon idell and Crucian carp proteins were distributed in the region of MW35-55kDa, pH7-5and MW40-55kDa, PH5-3. The protein expression difference between sea fish and river fish were distributed in the region of MW45-53kDa, pH5-3and MW35-52kDa, pH7-5.3. Compared different aspects of four fish, in terms of biochemical composition, textural properties, microstructure and so on in order to find connection with2-DE information The results showed that muscle fibers of river fish were arranged closer than sea fish muscle fibers. Muscle hardness of river fish was correspondingly higher. Abundance difference of river fish(Ctenopharyngodon idell and Crucian carp) and sea water fis (Pseudosciaena crocea and Lateolabrax japonicas) indicated the different deform degree of actomyosin (actin), thus resulting in different hardness properties of structure. At the same time, muscle elastic and cohesion properties of sea fish muscle were smaller than those of river fish. The proteins of river fish on2-DE, which were close to the20kDa, had greater abundance (especially Ctenopharyngodon idell). The color of sea fish muscles were more red and green than river fish muscle color due to containing a higher proportion of red muscle fibers, while river fish muscle color was more light and yellow blue than sea fish muscle. Because myoglobin molecular weight close to18kDa, so proteins which were close to20kDa might be related with myoglobin related proteins. Different kinds of microfiber structure of sea fish and river fish might be associated with actin and tropomyosin content. The proteins of sea fish and river fish on2-DE indicated that of the abundance of tropomyosin and actin proteins was significantly different.