| Scylla paramamosain(mud crab),belonging to the Crustacea,Scylla,is one of the main precious marine crab species aquacultured in the southeastern coastal areas of China.Based on the transcriptome data of S.paramamosain,this study is to clone and analyze the expression patterns of Na~+/H~+-exchanger,Na?/K?-ATPase and ANT2 related to regulation during the growth and development of Scylla pseudoacuminata and salinity stress from the perspective of molecular biology.The main results are as follows:1.Molecular cloning and expression analysis of the Na~+/H~+-exchanger in S.paramamosainNa~+/H~+-exchanger is a membrane-associated enzyme responsible for the active transport of Na~+and H~+ions across cell membranes and generating chemical and electrical gradients.It plays an important role in salinity adaptation process of aquatic crustacean.S.paramamosain is a mud crab commonly consumed in Southeast Asia,whose growth,development and immunity of the crab are significantly affected by salinity variation.In order to investigate the function of Na+/H+-exchanger in Scylla paramamosain under salinity stress,we obtained a full-length of JHEBP cDNA sequence from the mud crab,Scylla paramamosain.The full length of Na~+/H~+-exchanger cDNA was 3,624 bp,with a predicted 2886 bp open reading frame(ORF)encoding 961 amino acids with a predicted molecular weight of 107.1 kD.Comparison with homologous proteins showed that the deduced Na~+/H~+-exchanger sequence has the highest sequence identity to Portunus trituberculatus(84.9%),and the two sequences were clustered into one group by phylogenetic analysis.Typical domains including one signal peptide,one Na~+/H~+-exchanger domain and twelve transmembrane alpha helixes were found in amino acid sequence of Na~+/H~+-exchanger.Furthermore,the quantitative real-time polymerase chain reaction revealed that Na~+/H~+-exchanger mRNA was ubiquitously expressed in all of the detected tissues,with the highest expression in the ovary,followed by gill,testes and gill.During the larval development,the expression of Na~+/H~+-exchanger was highest in fertilized eggs,followed by megalopa stage.During salinity stress,the expression of Na~+/H~+-exchanger in low salinity groups(7 and 17)increased significantly during 20 min.Subsequently,the expression was down-regulated during 1h,then gradually rising and returning to normal during 2h-6h.The expression of Na~+/H~+-exchanger in high salinity group(37)was decline first and then rise gradually during 20min-2h,and the expression level significantly Significant decline during 6h.Taken all those things together,we hypothesized that Na~+/H~+-exchanger of Scylla paramamosain plays a vital role mainly in megalopae stage in the process of salinity adaptation.Low salt significantly induced the high expression of Na~+/H~+-exchanger gene.It is speculated that the Na~+/H~+-exchanger gene plays an important role in osmotic regulation in low salt environment,which is of great significance to the breeding of new low salt tolerant strains of marine crab and the improvement of the success rate of breeding.2.Molecular cloning and expression analysis of the Na?/K?-ATPase in S.paramamosainThe osmotic organs of crustaceans include gills,viscera and antennal glands(or excretory organs).Gill has many functions,including gas exchange,osmotic regulation,acid-base balance and nitrogen excretion.The crab is a kind of broad-salt sea crab,which can actively regulate the osmotic pressure of its hemolymph.Nitrogen and potassium triphosphatase are the main driving forces of ion transport.The activity of Na?/K?-ATPase reflects the osmotic regulation of crustaceans.The alpha subunit of Na?/K?-ATPase is a multimembrane protein with a molecular weight of about 110 kDa.The catalytic site of the enzyme located on the alpha subunit is responsible for transporting ions.According to the reported literatures,the full-length Na?/K?-ATPase gene of Scylla pseudoacuminata contains 3866 bp,encoding 1039 amino acids in a complete open reading frame.However,the expression of Na?/K?-ATPase in different tissues and different stages of Scylla pseudoacuminata has not been studied in detail.In this chapter,based on the predecessors,the bioinformatics analysis of Na?/K?-ATPase and its coding amino acid sequence was carried out.The amino acid sequence of Na?/K?-ATPase was compared with other crustaceans by Blast,and the expression changes of the gene in different tissues and larvae were analyzed by real-time quantitative PCR(RT-qPCR).At the same time,the expression of Na?/K?-ATPase was studied under salinity stress in the larvae of Macrobrachium pseudoacuminatum,which were more susceptible to salinity changes and directly affected the molting metamorphosis and survival rate of marine crabs.3.Molecular cloning and expression analysis of the ANT2 in S.paramamosainANT is an ADP/ATP Carrier(AAC),a member of the mitochondrial transporter superfamily.ANT,which accounts for about 12%of mitochondrial membrane proteins,is a hydrophobic protein.Its function is to transport adenosine triphosphate(ATP)synthesized in mitochondria to the cytoplasm,and transfer adenosine diphosphate(ADP)into the mitochondria to maintain mitochondrial metabolism and energy consumption through oxidative phosphorylation.ANT protein consists of two identical subunits of ATP/ADP binding site,which can realize the transmembrane exchange between ATP in mitochondria and ADP in cytoplasm through conformational changes.ANT2 is highly conservative in structure and function,and plays an important role in the process of apoptosis induction,cell proliferation related to mitochondrial diseases,and regulating the physiological process of cold tolerance in organisms.Although crabs are broad-temperature and salt crabs,the changes of temperature and salinity in different seasons affect their distribution and migration,and can affect their physiological and biochemical changes in vivo.Some studies have shown that ANT2 plays a role in salinity adaptation of S.paramamosain,but no relevant studies have been conducted on the expression of this gene in its larvae.In order to further elucidate the molecular mechanism of environmental adaptation of Scylla pseudoacuminata,this chapter,on the basis of predecessors,carries out bioinformatics analysis of ANT2 and its coding amino acid sequence,and analyses its amino acid sequence with Blast alignment.The homology of other crustaceans was analyzed by real-time quantitative polymerase chain reaction(RT-qPCR).At the same time,the expression of ANT2 under salinity stress was studied to explore the molecular mechanism of low-salt adaptation of Potentilla crab,and to provide theoretical guidance for the cultivation and management of Potentilla crab.In summary,we cloned two genes Na~+/H~+-exchanger,Na?/K?-ATPase and ANT2related to salinity stress.Then we did some bioinformatics analysis and studied their expression patterns in the crab.The results showed that Na~+/H~+-exchanger,Na?/K?-ATPase and ANT2 may be involved in the larval growth and development of Scylla paramamosain.These basic studies provide data support for further revealing the osmotic pressure regulation mechanism of crustaceans. |
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