Physicochemical Index Analysis Of Haemolymph And Cloning And Functional Study Of Na~+/H~+-exchanger And V-ATPase Genes In Swimming Crab (Portunus Tritubereulatus) Under Different Salinity

The swimming crab(Portunus trituberculatus) was a large marine economic crab species. In china, large-scale artificial breeding was carried out and achieved good economic benefits. Salinity is an important environmental factor of artificial breeding of P. trituberculatus. Growth, development and survival of P. trituberculatus was affected by salinity. Drastic changes of salinity brings huge risks to artificial breeding of P. trituberculatus.There was few research about the mechanism of salinity adaptation in P. trituberculatus. Research of osmoregulation type, osmoregulation ion and the material for osmotic adjustment process in haemolymph of P. trituberculatus was not conducted yet. Few gene functional studies of main Ion transporter was conducted. In order to provide a reference for the study of adaptation to salinity, aquaculture feeding, osmoregulation mechanism and so on in P. trituberculatus. Physicochemical index analysis of haemolymph and functional study of V-ATPase and Na+/H+-exchanger genes in P. trituberculatus under different salinity was carried out in this study. The specific research content is divided into four parts as follows: 1. Effect of salinity stress on the physical level in the haemolymph of P. trituberculatusSalinity stress on P. trituberculatus was carried to find out characteristics and regularity of serum osmolarity, ion concentration and serum biochemical indicators during the crab adapt to salinity change. Four salinity gradients(salinity 5、10、20、50) experimental groups and a control group of salinity 30 were set up in the experiment. The results showed that serum osmolarity of low salinity experimental groups declined and serum osmolarity of salinity 50 experimental group rose during 0-12 h. During 12-72 h, the serum osmolarity of all the experimental group reached to a steady state. Along with the rising of the salinity, D-value between serum osmolarity and medium osmolarity was reduced firstly and then got steady. The change of serum Cl–、Na+、K+ ion concentration was like as the change of serum osmolarity. D-value of Cl–、Na+ ion concentration between serum and medium was firstly reduced and then steady. D-value of K+ ion concentration rose at first, then declined and got steady in the end. The values of serum glucose(GLU) 、 triglyceride(TG) and total protein(TP) of the experimental groups declined firstly and then stable. The values of serum GLU、TG and TP of the control group were stable during 0-72 h. While the content of serum GLU、TG and TP of the experimental group was stable, the larger difference of the salinity between experimental and control group, the greater decline of the content of serum GLU 、 TG and TP. Maximum decline of serum GLU and TP content was more than the maximum decline of serum TG content. The values of serum GLU of experimental groups declined quickly during 0-9 h. The values of serum TG and TP declined during 0-12 h. The maximum decline of the value of serum TP was less than the maximum decline of the value of serum GLU in salinity 20 experimental group. The maximum decline of the value of serum TP was more than the maximum decline of the value of serum GLU in salinity 5、10、50 experimental group. The values of serum urea(UREA) of the experimental groups rose firstly and then stable, and were significantly higher than the control group(P>0.05). The value of serum UREA of salinity 5 experimental group was near with the value of serum UREA of salinity 50 experimental group. However, the values of serum TP of salinity 10 and 50 experimental groups were near with each other. The results indicates that P. trituberculatus is belong to hypertonic regulation crab. Cl– and Na+ ions play a key role in the process of osmolarity regulation. Serum GLU and TP of P. trituberculatus were main materials for energy supply and the serum GLU supplied energy firstly in the osmoregulation process. Partial content increase of serum free amino acid of P. trituberculatus could be derived from other tissues in high salt water environment. 2. Na+/H+-exchanger in P. trituberculatus: cloning, characterization, and mRNA expression under salinity stressIn order to investigate the function of Na+/H+-exchanger in P. trituberculatus under salinity stress. Na+/H+-exchanger cDNA(Gen Bank: KU519329) was cloned from gill tissue of P. trituberculatus by RACE(rapid-amplification of cDNA ends). The expression of Na+/H+-exchanger gene in different tissues and under the salinity stress was analysed by the RT-qPCR(Reverse transcription-qPCR). Analysis of the nucleotide sequence revealed that the full-length of Na+/H+-exchanger cDNA(Gen Bank: KU519329) was 4 233 bp(base pairs). ORF(open reading frame), 5’ UTR(untranslated region), and 3’ UTR were 2 961 bp, 519 bp and 753 bp respectively. ORF encoded 986 amino acids. Its calculated molecular weight was 110.8kDa. Biological information analysis showed that Na+/H+-exchanger amino acid sequence homology between P. trituberculatus and Carcinus maenas was the highest, reached 87.2% and there was a signal peptide and twelve transmembrane alpha helixes. Phylogenetic analysis showed the closest relationship was between P. trituberculatus and C. maenas. Analysis of RT-q PCR showed P. trituberculatus Na+/H+-exchanger expression level was highest in gill. During salinity stress, Na+/H+-exchanger expression levels of P. trituberculatus gill tissue in control group and 50 salinity experimental group were basically stable. On the whole, Na+/H+-exchanger expression levels of gill in 5, 10, and 20 salinity experimental group increased significantly at 0-12 h, and the gene expression was down-regulated during 24-168 h. These results indicated that P. trituberculatus belongs to the "weak" hyperosmotic regulation crab, Na+/H+-exchanger plays a vital role mainly in low salt conditions in the process of salinity adaptation. 3. V-ATPase subunit d in P. trituberculatus: cloning, prokaryotic expression and mRNA expression under salinity stressIn order to investigate the function of V-ATPase subunit d in P. trituberculatus under salinity stress. V-ATPase subunit d cDNA was cloned from gill tissue of P. trituberculatus by RACE. The expression of V-ATPase subunit d gene in different tissues and under the salinity stress was analysed by the RT-qPCR. Analysis of the nucleotide sequence revealed that the full-length of V-ATPase subunit d cDNA(Gen Bank: KU519330) was 2 307 bp, ORF, 5’ UTR, and 3’ UTR were 1 047 bp, 96 bp and 1 164 bp respectively. ORF encoded 348 amino acids. Its calculated molecular weight was 39.7k Da. Biological information analysis showed that V-ATPase subunit d amino acid sequence homology between P. trituberculatus and Melipona quadrifasciata was the highest, reached 83% and there was no signal peptide and the sequence was highly conservative. Phylogenetic analysis showed the closest relationship was between P. trituberculatus and M. quadrifasciata. Analysis of RT-qPCR showed P. trituberculatus V-ATPase subunit d expression level was highest in gill. During salinity stress, V-ATPase subunit d expression levels of P. trituberculatus gill tissue in control group was basically stable during 0-168 h. V-ATPase subunit d expression levels of gill in 5, 10, and 20 salinity experimental group increased significantly at 0-12 h, and the gene expression was down-regulated during 24-168 h. The expression level of gill in 50 salinity increased slightly during 0-9 h and its level was down-regulated during 12-168 h. Analysis of proteomic Mass Spectrom showed peptide of expression on peptide of theory cover reached 32%. These results indicated that V-ATPase subunit d plays a vital role mainly in low salt conditions and a weak effect in high salinnity in the process of salinity adaptation. V-ATPase subunit d fusion protein construction was completed. 4. Cloning of V-ATPase subunit a in P. trituberculatus and effect of salinity stress on mRNA expression of V-ATPase subunit a and Na+/K+-ATPase subunit α and activity of V-ATPase and Na+/K+-ATPaseIn order to investigate the effect of salinity stress on mRNA expression of V-ATPase subunit a and Na+/K+-ATPase subunit α and activity of V-ATPase and Na+/K+-ATPase in P. trituberculatus. V-ATPase subunit a cDNA was cloned from gill tissue of P. trituberculatus by RACE. The expression of V-ATPase subunit a gene in different tissues and under the salinity stress was analysed by the RT-qPCR. The activity of V-ATPase and Na+/K+-ATPase was determined by kit. Analysis of the nucleotide sequence revealed that the full-length of V-ATPase subunit a cDNA(GenBank: KU519328) was 3 167 bp, ORF, 5’ UTR, and 3’ UTR were 2 484 bp, 174 bp and 509 bp respectively. ORF encoded 827 amino acids. Its calculated molecular weight was 95 kDa. Biological information analysis showed that V-ATPase subunit a amino acid sequence homology between P. trituberculatus and L. vannamei was the highest, reached 77.6% and there were six transmembrane domains. Phylogenetic analysis showed the closest relationship was between P. trituberculatus and L. vannamei. Analysis of RT-qPCR showed P. trituberculatus V-ATPase subunit a expression level was highest in gill. During salinity stress, V-ATPase subunit a expression levels of P. trituberculatus gill tissue in control group was basically stable during 0-168 h. V-ATPase subunit a expression levels of gill in 5, 10, and 20 salinity experimental group increased significantly at 0-12 h, and the gene expression was down-regulated during 24-168 h. The expression level of gill in 50 salinity increased slightly during 0-12 h and its level was down-regulated during 24-168 h. Analysis of RT-qPCR showed P. trituberculatus Na+/K+-ATPase subunit α expression level was highest in gill. During salinity stress, Na+/K+-ATPase subunit α expression levels of P. trituberculatus gill tissue in control group was basically stable during 0-168 h. Na+/K+-ATPase subunit α expression of gill in 5, 10, and 20 salinity experimental group was up-regulated at 0-12 h, and the gene expression was down-regulated during 24-168 h. The expression level of gill in 50 salinity increased slightly during 0-12 h and its level was down-regulated during 24-168 h. Analysis of enzymatic activity showed that enzymatic activity in control group and 50 salinity experimental group of Na+/K+-ATPase and V-ATPase control group was basically stable during 0-168 h. The activity of Na+/K+-ATPase and V-ATPase in 5, 10, and 20 salinity experimental group rose at first, then declined and got steady in the end. Activity of V-ATPase in 50 salinity experimental group was up-regulated during 9-12 h and 12-168 h declined and then got steady. These results indicated that Na+/K+-ATPase plays a role mainly in low salt conditions and no effect in high salinity in the osmoregulation process. V-ATPase plays a role in osmotic adjustment in adaptation of low salt and high salt.