Identification Of The Aquaporins And Na~+/K~+-ATPase Genes In Pacific Abalone,Haliotis Discus Hannai And Analysis Of The Responses To Salinity Changes

As one of the most important commercially fishery industry animal,Pacific abalone Haliotis discus hannai was widely cultured in northern China.At present,the response to osmotic change in Pacific abalone has not been studied systematically.In this study,the related genes involved in osmotic pressure regulation in Pacific abalone were studied.It has important theoretical guiding significance to production and practice.Aquaporin(AQP)mediated the transmembrane transport of water molecule.In addition,it plays a crucial role in the regulation of osmotic pressure.In this study,we identified 18 AQPs from Pacific abalone genome using bioinformatics combined with molecular biology.The accurate sequences were confirmed by sanger sequencing.Cluster analysis indicated that the 18 AQPs could be clearly divided into 4 clusters include all the 4 subfamilies named C-AQP,AQP-8,AQGP and S-AQP.According to their subfamilies,they were respectively named HdAQPc1-c9,HdAQP8.1-8.4,HdAQPg1-g3 and HdAQPs1-s2.Multiple alignment analysis shown that the 18 amino acid sequences were highly conserved in the two NPA motifs.Comparison of the amino acids in the Ar/R region shown that in the C-AQP subfamily,the first two sites were Phe and His in general.Hole analysis shown that the diameter of the AQPs in this subfamily was about 1.6?.But the Ar/R amino acids component in HdAQPc3,HdAQPc8 and HdAQPc9 were very special.In HdAQPc3,both of the first two amino acids were replaced by Ala.In HdAQPc8 and HdAQPc9,the second amino acid was replaced by Ile.Hole analysis shown that the replacements result in a wider diameter than the others in this subfamily.While the functional analysis shown that only HdAQPc9 possess the ability for glycerin transport.HdAQPc3 and HdAQPc8 may possess the ability for the other compounds transport.In addition,the fourth site of the Ar/R region in HdAQPc4 replaced by Trp.This replacement may enable HdAQPc4 to transport other osmotic effectors and play the role of osmotic pressure regulation.In AQP-8 subfamily,His usually appeared at the first site of the Ar/R region.In general,this region of the AQP-8 subfamily does not contain hydrophobic amino acids.Functional analysis show that the stain expressing HdAQP8.2 could survived at the hypertonic condition.This pattern indicated that the AQP inability to transport glycerin.Moreover,this stain also grew well at the hypotonic condition.This pattern indicated that glycerol is not the only osmotic effector in yeast.Additionally,HdAQP8.2 mediated the transport of these solutes.In AQGP subfamily,the first two site of the Ar/R region are usually Gly,the smallest?amino acid.This lead the hole diameter of the AQPs in this subfamily relatively wider.While functional analysis indicated that only HdAQPg1 could mediated the transport of glycerin.These data suggested that the other two AQPs in this subfamily may mediate the transport of other macromolecular substances.Detection of the 18 AQPs expression in different tissues of Pacific abalone after high or low salinity treatments shown that the response patterns of each AQPs to the same salinity change were diverse in different tissues,and the response patterns of each AQPs to different salinity change were also diverse in the same tissue.These data indicate that the maintenance of osmotic pressure balance is the result of multiple AQPs participating and coordinating with each other.Moreover,it also reflects the diversity of osmotic pressure effectors in the Pacific abalone.As an ion transporter,Na~+/K~+-ATPase(NKA)could driving 3 Na~+export and 2K~+import across the plasma membrane against their electrochemical gradients using the energy derived from ATP hydrolysis.In the present study,we have cloned and characterized the full-length cDNAs of NKA?subunit and?subunit from Pacific abalone.Multiple alignment shown that the predicted protein sequence of the NKA?subunit,as the catalytic subunit,was well conserved(73.55%-89.24%).Moreover,the highly conserved regions were consistent with the transmembrane regions.In contrast,the protein sequence of the?subunit had low similarity with those of other species(23.96%-47.46%).When treated the Pacific abalones with sudden salinity change(high or low salinity),the concentration of ions in hemolymph increased or decreased rapidly.While in tissues,the concentration of ions was maintained within the normal range.These data suggested that Pacific abalone has a strong osmotic regulation ability when faced with salinity change.The mRNA expression levels of NKA?and?subunits in tissues were detected after the sudden salinity change treatment.From these data we could conclude that at the initial stage,high salinity treatment led to a small decrease in both subunits and then a large increase.On the contrary,both subunits showed a small increase,but then a large decrease after the low salinity treatment.Meanwhile,the protein expression trend of NKA?subunit and its activity in tissues were measured,which was consistent with the mRNA expression pattern.Finally,the concentration of cAMP in tissue after sudden salinity change were measured.These data indicated that the high salinity treatment leads the increase of cAMP concentration.In contrast,low salinity treatment leads the decrease of cAMP concentration.Correlation analysis shown that the concentration of cAMP was positive correlated with the NKA mRNA expression and its activity.Therefore,NKA played an important role in the process of osmotic regulation in Pacific abalone,and its expression was regulated by cAMP.