| Chinese sturgeon (Acipenser sinensis) is a rare species and endemic to China. So far, domestic scholars have done some helpful studies on osmolality of juvenile Chinese sturgeon, but it can't meet the need of protection of Chinese sturgeon. Hence, the NKA gene sequence and expression, location of iron transport proteins in gills of juvenile Chinese sturgeon were studied for the object of molecular mechanism in osmolality in this study.In order to establishment of nonlethal biopsy method on hybrid sturgeon, we examined the changes in growth indexes, osmolality, irons concentration and NKA activity among three group (nonlethal biopsy group, NB, control group, CG, fresh water group, FW) both at the beginning and the end of experiment. The results indicated that:the growth indexes, osmolality, irons concentration and NKA activity of hybrid sturgeons between in NB group and control group did not significant change (P> 0.05). Compared with FW group, the growth indexes of sturgeons in NB group under the condition of salinity 10 did not show remarkable difference, but it's osmolality, irons concentration and NKA activity were obviously higher (P< 0.05) than that in FW group. The results suggest that the sturgeons sampled by nonlethal biopsy method can regulate osmolality in balance.To explore the molecular regulation mechanism of Na+, K+-ATPase (NKA) a-subunit in gills of juvenile Chinese sturgeon on salinity 10, Partial NKA a-subunit gene sequence was cloned, and compared with other species of fish. The results showed this sequence was highly conservative in different fish. The abundance of a-subunit mRNA, NKA activity, iron concentration and osmolality were examined at 3,6,12,24,48,72,96 hour post-transfer. Results showed that:In experimental group (fresh water-salinity water, FW-SW), the expression of NKA a-subunit mRNA and NKA activity increased significantly (P<0.05) during the adaptation phase (0-12 h), and the concentration of Na+, Cl- and osmolality rose to a maximum level at 12 h post-transfer. In regulatory phase (12-24 h), the amounts of NKA a-subunit mRNA and NKA activity fell to a relatively lower level which still higher than those of fish in control group (P< 0.05). The change both in iron concentration and osmolality were in accordance with the change of NKA and mRNA expression. In conclusion, gill Na+, K+-ATPase in Chinese sturgeon plays an important role in osmolality regulation by changing a-subunit mRNA expression. The different a-subunit mRNA expression can lead to the variation of enzyme activity which can further regulate the iron concentration and osmolality in balance.For the purpose of studying the tissue distribution of NKA and NKCC in gills from juvenile Chinese sturgeons, the immunoreactive cells were examined by immunocytochemical approach. The tests revealed that:NKA-positive cells were widely present in the filament and lamellae. The number and staining intensity of NKA-positive cells in salinity 10 group was more than that of fresh water group. Moreover, NKA-positive cells distributed in cytoplasm except nucleus. On the other hand, the distribution of NKCC-positive staining cells was in lamellae. The numbers of NKCC-positive cells between salinity 10 group and fresh water group were not dramatically different. However, the NKCC-positive cell shape was changed because of salinity. The intensity of immunofluorescent in salinity 10 group was slightly higher than that of fresh water group. Meanwhile, NKCC-positive cells showed convex folds surfaces. The distribution of NKCC staining was throughout the cell and NKCC staining intensity was less bright than NKA staining. This difference indicated the less protein abundance in NKCC-positive cells. In addition, the distribution of NKA staining cells was different from that of NKCC staining cells. In conclusion, Salinity effects on the distribution of branchial NKA-immunoreactive and NKCC immunoreactive. |
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