| Eriocheir sinensis,commonly known as river crab,hairy crab,hairy crab and clear water crab,is a famous freshwater crab in China.It is large,nutritious,delicious meat with high economic value.Eriocheir sinensis mainly distributes in the middle and lower reaches of the Yangtze River and coastal provinces.It has the habit of reproductive migration.At the turn of autumn and winter every year,sexually mature individuals swim along the river to the saltwater water confluence area where the estuary enters the sea to mate and lay eggs.Larvae develop into juvenile crabs in saltwater water.Juvenile crabs grow up in saltwater water.After 5-6 times of molting,adult crabs continue to grow and fatten in freshwater.During the migration of Eriocheir sinensis,great changes have taken place in environmental salinity,which has great influence on gonad maturation and reproductive migration of Eriocheir sinensis.Eriocheir sinensis can adapt to the changes of external salinity by its special osmotic pressure regulation mechanism.In order to better understand the physiological mechanism of osmotic pressure regulation in Eriocheir sinensis,this study used comparative transcriptome and comparative proteomics techniques to preliminarily explore the gene transcription level expression and eggs of the gill tissue of Eriocheir sinensis male adult crabs under short-term high salinity stress.The biological processes of energy metabolism,information transduction,ion transport,immune stress,amino acid metabolism,fatty acid metabolism and cytoskeleton protein in the hind gill tissue of Eriocheir sinensis under high salinity stress were comprehensively analyzed.Based on transcriptome studies,important ion channel proteins associated with osmotic pressure regulation of Eriocheir sinensis,sodium,potassium and chloride,were identified for co-transporting eggs.The full length of NKCC gene was obtained by cloning and its expression in different tissues,salinity and salinity stress time of Eriocheir sinensis was analyzed.1.Comparative transcriptionomic analysis of the posterior gill of Eriocheir sinensis under acute high salinity stressTo reveal the genes and pathways involved in osmoregulation,adult male crabs?body weight=110±5 g?were acclimated for 144 h in freshwater?FW,0 ppt?or seawater?SW,25 ppt?.Changes in the transcriptome of crab gills were then analysed by RNA-Seq,and 174,903unigenes were obtained.Comparison of genes between FW-SW-acclimated groups identified 932 genes that were significantly differentially expressed in the gill,comprising 433 and 499 up-and downregulated transcripts.Gene Ontology functional enrichment analysis revealed that important biological processes related to salt stress were significantly enriched,including energy metabolism,ion transport,signal transduction and antioxidant activity.Kyoto Encyclopaedia of Genes and Genomes enrichment analysis mapped the differentially expressed genes to241 specific metabolic pathways,and pathways related to energy metabolism,oxidative phosphorylation and the tricarboxylic acid?TCA?/citrate cycle were significantly enriched.Salinity stress altered the expression of many enzymes involved in energy metabolism,ion transport,signal transduction and antioxidant pathways,including citrate synthase?CS?,Na+/K+-ATPase?NKA?,NKCC dopamine receptor D1?DRD1?,synaptic binding protein 1?STXBP1?,Cu2+/Zn2+superoxide dismutase?SOD1?and glutathione S-transferase?GST?.Additionally,the obtained transcriptomic sequencing data provides a useful resource for identification of novel genes,and further physiological analysis of Chinese mitten crab.2.Comparative proteomic analysis of the posterior gill of Eriocheir sinensis under acute high salinity stressIn experiment 1,transcriptome technique was used to compare the expression of all genes in gill cells of Eriocheir sinensis under high salinity stress.Protein is the main undertaker of cell function.Proteome can directly describe the cell function and state under specific physiological conditions.Therefore,on the basis of experiment 1,Experiment 2 analyzed the changes of protein expression in gill of Eriocheir sinensis after high salinity stress.A total of 1453 proteins were identified by label free proteomics.Under the threshold of 2-fold differential multiple?FC?,242differential proteins were screened,including 122 up-regulated differential proteins and 120 down-regulated differential proteins.GO database and KEGG database were used to annotate and enrich differential proteins.It was found that differential proteins were significantly enriched in energy metabolism,signal transduction,ion transport,actin cytoskeleton,immunity,lipid metabolism,amino acid metabolism and other biological functions.The results showed that the energy metabolism of Eriocheir sinensis decreased and the expression of actin and cytoskeleton protein increased after 144 hours of high salinity stress.Under high salinity stress,Eriocheir sinensis responds to oxidative damage caused by high salinity by improving immunity and antioxidant capacity.3.Cloning and expression analysis of NKCC gene from Eriocheir sinensisNKCC is a SLC12A family protein that transports Na+,K+,and Cl-into cells,and plays an important role in ion transport and osmotic regulation in aquatic animal cells.In this study,NKCC gene was cloned from the hind gill of Eriocheir sinensis by RT-PCR and RACE.The full length of NKCC gene was 4127 bp,of which the length of 5'non-coding region?UTR?was18 bp,the length of 3'non-coding region was 944 bp,and the length of open reading frame was 3165 bp,encoding 1054 amino acids.Physicochemical analysis predicted that its molecular weight was 51.066kD and its theoretical isoelectric point was 4.65.The secondary structure of NKCC was predicted to consist of 10 transmembrane domains.The homologous comparison showed that the amino acid sequence of NKCC was the most similar to that of NKCC of Hawaiian cave shrimp,with a similarity of 77%.Phylogenetic analysis showed that NKCC was clustered with NKCC of Hawaiian marine cave shrimp,blue crab and other crustaceans.Real-time fluorescence quantitative analysis?qRT-PCR?showed that NKCC was expressed in gill,hepatopancreas,stomach,heart,intestine,serum,eyestalk,thoracic nerve node and brain nerve node of Eriocheir sinensis,and the expression of NKCC in intestine of Eriocheir sinensis was the highest.The expression of NKCC in the intestine of Eriocheir sinensis changed significantly with the prolongation of stress time under acute salinity of 25,and restored to a stable level after 72 hours of stress,suggesting that NKCC may be involved in osmotic pressure regulation of Eriocheir sinensis and play an important role in osmotic pressure balance of Eriocheir sinensis.The results provide a theoretical basis for further study on the osmotic regulation mechanism of ion channels in Eriocheir sinensis. |
PDF Full Text Request