Cloning And Expression Of Four Aquaporin Homologs From The Chinese Black Sleeper(Bostrychus Sinensis):The Effects Of Salinity Acclimation

The Chinese black sleeper(Bostrychus sinensis)is a euryhaline fish,which prefers to inhabit in foreshore and estuaries areas,and has strong osmoregulatory capacity.Aquaporins of fishes play a key role in maintaining osmotic pressure balance.To gain a better understanding of the role of aquaporins in osmoregulatory of Chinese black sleeper,the coding sequences of aqp1 a,aqp3a,aqp8 a,and aqp9 a were cloned for the first time.The tissue expression patterns and transcript abundance of aqp1 a,aqp3a,aqp8 a,and aqp9 a in Chinese black sleeper that were acclimated to salinity of 0(freshwater),5,15,and 40 ppt for 30 days were measured by real-time quantitative polymerase chain reaction(RT-PCR).The results showed that the coding DNA sequence of Chinese black sleeper aqp1 a spanned 786 bp in length and encoded 261 amino acids.The coding DNA sequence of aqp8 a spanned 795 bp in length and encoded 264 amino acids.Both coding DNA sequence of aqp3 a and aqp9 a spanned 915 bp in length and encoded 304 amino acids.The four aquaporins of Chinese black sleeper have many features that were consistent with this protein family that found in other species,including two NPA motifs located in the second and fifth loops,five connecting loops,and cytoplasmic N-and C-terminal domains.The protein transmembrane domain prediction showed that Aqp1 a,Aqp8a,and Aqp9 a had six transmembrane domains,while Aqp3 a had seven transmembrane domains.The Chinese black sleeper aqp1 a,aqp3a,aqp8 a,and aqp9 a mRNAs were expressed in the heart,brain,testis,spleen,stomach,skin,gill,intestines and eyes.The expression of aqp1 a mRNA in eyes was the highest,followed by intestines,skin and gills.The expression of aqp9 a mRNA was the highest in eyes,followed by gills and testis,while the expression in skin,stomach,intestine and spleen tissues was low.The expressions of aqp3 a mRNA and aqp8 a mRNA was highest in gills and intestine.The expression of aqp1 a,aqp3a,and aqp9 a in the gills increased significantly in the fish transferred to freshwater and 5 ppt,whereas the expression of aqp8 a decreased significantly.The expression of aqp1 a and aqp9 a in gills of Chinese black sleeper did not change significantly when the fish were transferred to 40 ppt,but the expression of aqp8 a increased significantly.In the Chinese black sleeper intestine,the expression of aqp1 a,aqp3a,and aqp8 a increased significantly in fish acclimated in 40 ppt,while the expressions of them decreased significantly in the 5 ppt seawater.The expression of aqp1 a,aqp3a,aqp8 a,and aqp9 a in gills and intestine of Chinese black sleeper also changed during a short period(0 h-48 h)of salinity adaptation.These findings suggested that aqp1 a,aqp3a,and aqp9 a may play a role in water transport in the gills in the freshwater or 5 ppt seawater acclimated Chinese black sleeper.The aqp1 a,aqp3a,and aqp8 a were involved in water transport in the intestine in the 40 ppt seawater acclimated Chinese black sleeper.