| The molecular osmoregulation mechanism of the euryhaline fish is a hot topic of study of fish physiology at present. Anguilla marmorata is the typical catadromous migration fish in China. Glass eels must migrate from seawater to freshwater for growth and development,but we do not know that the molecular mechanism in the elvers of Anguilla marmorata reponse to salinity adaptation during different the variation of salinity, and the basic research in this area is also delayed. So, the gene cloning, mRNA expression, protein expression, enzyme activity and location of irontransport proteins in gill of juvenile Anguilla marmorata were Studied for the object of molecular mechanism in osmolality in this study, and study mainly around on the regulation mechanism of juvenile Anguilla marmorata Vacuolar-Type-H+-ATPase(VHA), Na+/K+-ATPase(NKA) and FXYD11 adapt to salinity change. To further anlysis the different expression of regulation gene at the mRNA level and protein level and its important effect on acclimating the salinity variation, the study aims to reveal the regulatory mechanism of the elvers to adapt to salinity variation. The results are as follows:(1)The Vacuolar-Type-H+-ATPase β1(VHAβ1) plays an important role in osmoregulation of fish. In the present work, the full-length cDNA of VHAβ1 (named Am VHAβ1)with 1741 base pairs (bp) was identified in Anguilla marmorata.It contained a 1512 bp open reading frame encoding a polypeptide with 503 amino acids, a 83 bp 5’-untranslated region (UTR) and a 146 bp 3’-UTR. Sequence alignment showed that AmVHAβ1 shared 91-99% identity and identical structural features with its counterparts reported in other animals. Meanwhile, we first identified Na+/K+-ATP ase al (AmNKAal) and FXYD11 (AmFXYDll) in Anguilla marmorata. The full length cDN A of AmNKAal consists of 3,237 base pairs (bp), including a 3,069-bp open reading frame (ORF) that encodes an 1,022-amino acid polypeptide. The full length cDNA of AmFXYD11 is 739 bp, and contains a 213-bp ORF encoding a 70-amino acid polypeptide. AmFXYDll contains an ATP1G1_PLM_MAT8 domain, which is a regulatory structure ofNa+/K+-ATPase.(2) The expression levels of AmVHAβ1 in gill and kidney of A. marmorata were evaluated at different time points (0,1,3,6,12,24,48,72 and 96 hours as well as 15 days) during the exposure to salinity (0‰,10‰ and 25‰). The results indicated that the expression levels of VHAβ1 mRNA in gill and kidney revealed a significant increase and reached the highest level at 1 hour in brackish water (BW,10%o) group and 6 hours in seawater (SW,25%o) group. Therefore, the salinity could affect the relative expression level of AmVHAβ1 mRNAin gill, which exhibited the enhancement by approximately 44 times in SW group when compared with that in fresh water. What’s more, no remarkable difference in the expression of AmVHAβ1 mRNAwas observed within 15 days of seawater exposure (P> 0.05). Meanwhile, we evaluated the expression and activity of NKA and the mRNA expression of FXYDll in gill and kidney of A. marmorata at different time points (0,1,3,6,12,24,48,72,96 and 360 hours) upon salinity exposure at 0‰,10%o and 25%o. The expression of NKAalas well as mRNA expression of AmFXYD11 in gill were higher in brackish water (BW) eel and sea water (SW) eel than in fresh water (FW) eel (P<0.01), mRNA expression of NKAal and FXYD11 in gill almost at the same time reached the highest expression level. The expression pattern of FXYD11 gene in elvers gills are similar to the expression pattern of NKAα1. What’s more, the change trend of gill FXYD11 and NKAal mRNA expression are the same. But the amount expression of renal FXYD11 in the control group,10 and 25 salinity is at the same level. The results show that elvers in gill NKA alpha sub unit related to FXYD11 and no correlationin the kidney. However, the level ofNKAexpression inkidney of BW-acclimated eel was higher thanthat of SW- and FW-acclimated eel. Conversely, there was no significant difference of AmFXYD11 expression in renal between the two groups. The level of AmFXYDll expression in kidney was higher in FW group than in BW/SW group.(3) Immunoblotting analysis showed that VHA expression was significantly higher in BW and SW group, and the highest expression level of VHA protein in gill was detected at 96 h exposure in BW and SW group. V-H+-ATPase activity exhibited an increase by 2-3% in gill and kidney. The consequence primarily confirmed that Am VHAβ1 gene in elvers from A. Marmorata plays an important role in adaptation response to seawater. The protein expressionand activityofNKA in gill were higher in brackish water (BW) eel and sea water (SW) eel than in fresh water (FW) eel (P<0.01), and the highest protein abundance and specific activity of gill NKA in BW and SW were also detected later than the highest mRNA expression level. These results clearly demonstrate the AmNKAal and AmFXYD11 paly important roles of regulating in osmotic ho meo stasis of juvenile A. marmorata under saline environment.
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