The Effects Of Hunger On The Trypsin Activity And Its MRNA Expression Level Of Juvenile Lateolabrax Japonicus

Effect of hunger on digestive enzymes activities of Pisces has appealed intensively and extensively to increasing number of researchers throughout the academic world. Combining the traditional Enzymology methods with modern molecular biology techniques, we have examined the trypsin activity of juvenile Lateolabrax japonicus under starvation status and under further refeed status, moreover we have examined how hunger play important role in trypsin mRNA expression.The juvenile Lateolabrax japonicus have been artificially cultured at 15.00 salinity for short terms before hunger test. Then we divided them into two groups, one was under starvation status while the other was given food normally as control, and each group has been further divided into two parallel groups respectively. Hunger group was re-feed again after 6 days'starvation whereas normal group was given food when the test began. We sample the hunger group at 0 day, 3th day, 6th day during in starvation and at 3th day, 10th day, 17th day during re-feed term; at the same time the normal group was sampled at 0 day, 3th day, 6th day, 9th day, 16th day , 23th day.The activities of the pepsin and trypsin of juvenile Lateolabrax japonicus were examined during starvation and refeeding. The results showed that the activities of the pepsin of the juvenile fishes that were starved 3, 6 days and were refed 3 days were higher than the feeding group, and the activities of the trypsin were lower than the feeding group; but the activities of the pepsin of the juvenile fishes that were refed 10, 17 days were lower than the feeding group, and the activities of the trypsin were higher than the feeding group. It indicated that the two proteinic enzymes of the starved group were significantly different from the feeding group.The total RNA of Lateolabrax japonicus was extracted with TRIZOL, and it's cDNA was obtained by AMV reverse transcription systems. We have amplified the cDNA sequence of trypsin of juvenile Lateolabrax japonicus, a DNA fragment of 535 base pairs with our own two primers designed based on the homologous sequence of several relative fish, which has already been recorded in Genebank. The similarity of these relatives has been compared by special software. We found that the Dicentrarchus labrax shares the counterpart of trypsin of the most similarity of Lateolabrax japonicus; about 92.71% of mRNA and 94.94% of the amino acids are identical.Fluorescent quantitation PCR has been applied to examine the mRNA transcription level changes of trypsin of juvenile Lateolabrax japonicus, whereby we learn of the synthesis and secretion of various digestive enzymes. The results indicated: that mRNA of trypsin of feeding group transcribe in high level at first and descend later, descend remarkably at 3th day and keep low level always during late stage; and that the general trend of trypsin mRNA changes of hunger group resemble that of feeding group. Under starvation at 3th day and 6th day, the average transcription level of trypsin of hunger group was lower than that of feeding group, and whereas at 3th day and 10th day during re-feed stage the reverse happened, the average transcription level of trypsin of hunger group was higher than that of feeding group. This truth is a little different from the traditional enzyme assay result. There are marked difference between the trypsin activities of hunger group and feeding group.From the result we can ascertain that starvation exerts effects both on trypsin activity and on its secretion. Starvation could mute the activity of trypsin and re-feed could stimulate trypsin to excrete remarkably.Though the result of fluorescent quantitation PCR was basically consistent with that of traditional Enzyme assays, a little diverge existing at 3th day during re-feed stage revealed that the changes in trypsin activity is not only determined by it's secretory volume , but also involved in the external circumstance the trypsin exist in.