| Salinity is one of the most important environmental factors affecting the survival,development,reproduction,growth and physiological functions of teleost fish,it will change due to natural factors or human factors,thus causing osmoti adjustment stress on fish and affecting its normal growth.Cobia,Rachycentron canadum,is a euryhaline fish with high market value.According to reports,the suitable salinity range of the adult R.canadum is between 22.5 and 44.5 ppt,but little is known about the suitable salinity range of its juveniles and its potential salinity adaptation mechanism.In view of this,this study uses juvenile R.canadum(Full length 13.70±0.91cm,weight 9.74±0.85g)as experimental material to study the effect of salinity on survival,growth,physiological and biochemical indicators and tissue structure of juvenile R.canadum,at the same time,through comparative transcriptome analysis,screening and identification of differentially expressed genes related to osmotic pressure regulation,and excavating SNP sites related to salinity adaptation.Finally,cloning the cDNA sequences of aquaporin AQP1 and AQP3 genes related to fish salinity adaptation,and explore the AQP1 and AQP3 genes under different salinity conditions using Real-time Polymerase Chain Reaction(RT-PCR)technology changes in expression levels in different tissues.The research results obtained in the paper are summarized as follows:1.Effects of salinity on survival and growth of juvenile R.canadum.Under the condition of water temperature of 26.5-28°C and dissolved oxygen greater than 6 mg/L,according to the two-point method,the salinity ensuring mortality of 70%for 30 d as a period of experiment was chosen as the survival critical salinity,while the salinity in the group with 70%as much as the highest growth rate was set to be the growth-suitable critical salinity.Based on these critical salinities,the optimum salinity suitable for the juvenile of R.canadum was studied.Salinities at which the highest survival or growth rates among groups were not significantly different by multiple comparison were set to be the optimal survival or growth salinities.After statistical analysis,the suitable survival salinity of juvenile of R.canadum(Full length 13.70±0.91cm,weight 9.74±0.85g)was 3.68-37.43,and the optimal survival salinity ranged from 10 to 34.The suitable growth salinity is 5.91 to37.15,and the optimum growth salinity is 22 to 31.2.Effects of salinity on serum osmolality,ion concentration and NKA enzyme activity of juvenile cobia.Taking the normal seawater salinity of 30 as the control group,10 salinity is the low salinity group,35 salinity is the high salinity group,after thirty days breeding experiment,it was found that the influence of salinity on the serum osmolality,Na~+and Cl~-concentration of juvenile R.canadum had similar trends.The serum osmotic pressure of the low-salinity group began to decline sharply on the first day and reached the lowest point the next day,and then gradually increased and stabilized,but it was still significantly lower than that of the control group(P<0.05).The serum osmotic pressure of the high-salinity group began to increase on the first day and began to decrease after reaching the highest point on the second day,which was significantly different from the control group and the low-salinity group in each time(P<0.05).The Na~+concentration of the low salinity group reached the minimum on the second day,and then gradually increased and stabilized.The Na~+concentration at each time was significantly higher than that of the control group(P<0.05).The Na~+concentration of the high salinity group reached the maximum on the second day,and then gradually decreased.There was no significant difference compared with the control group at the 30th day(P>0.05),but there were significant differences at other time(P<0.05).The Cl~-concentration of the low-salinity group reached the minimum on the 2nd day,then gradually increased and tended to be gentle.On the15th and 30th days,the Cl~-concentration in the serum had no significant difference compared with the control group(P>0.05).The Cl~-concentration in the high salinity group gradually decreased and stabilized after reaching the maximum on the 2nd day,which was significantly different from the control group and the low salinity group(P<0.05).The effect of salinity on Na~+/K~+-ATPase activity of R.canadum is more obvious.The low salinity group and the high salinity group were significantly different from the control group at each time(P<0.05),and the Na~+/K~+-ATPase activity was at the 2nd day in both the low salinity group and the high salinity group.After reaching the maximum value,it gradually drops and stabilizes.3.Effects of salinity on the tissue structure of juvenile cobia.With normal seawater salinity 30 as the control group,10 salinity as the low salinity group,and 35 salinity as the high salinity group,a 30-day culture experiment was conducted on juvenile R.canadum.After low-salinity adaptation,the widths of gill filaments and gill lamellae increased,the gap between gill lamellae decreased,and the number of chlorinated cells decreased.After high-salinity adaptation,the width of gill filaments and gill lamellae decreased,the distance between gill lamellae increased,and the number of chlorinated cells increased.The diameters of renal tubules at all levels in the low-salinity group increased,and the gap with the inner wall of the renal capsule was smaller than that of the control group.The glomerular atrophy in the high-salinity group was larger than the inner wall of the renal capsule in the control group.The thickness of monolayer columnar epithelium in the low salinity group increased,and the number of goblet cells decreased,while the cell body diameter of the goblet cells in the high salinity group increased,and there was no significant change in other aspects.4.Transcriptome analysis of salinity adaptation of juvenile cobia.The normal seawater salinity was 30 as the control group,10 salinity was the low salinity group,and 35 salinity was the high salinity group.After 30 days of breeding experiments,the gill,kidney,and intestine tissues of the R.canadum were sequenced and analyzed by transcriptome sequencing technology.As a result,3,95,080,114 clean reads were generated and then assembled into 65,318 unigenes with an N50 size of2,758 bp.There were 20,671 significantly differentially expressed genes(DEGs)included 8,805 genes adapted to hypo-salinity and 11,866 genes adapted to hyper-salinity.These DEGs were highly represented in steroid biosynthesis,unsaturated fatty acids metabolism,glutathione metabolism,energy metabolism,osmoregulation and immune response.The identified differentially expressed genes provide valuable information for studying the molecular mechanism of cobia salinity adaptation.Through the interaction of various genes and pathways,the level of transcription of the juvenile fish body changes,controls the response to changes in salinity,regulates other cell functions,and responds to changes in salinity while also determining salinity adaptive molecular strategy.5.Mining and analysis of SNPs related to salinity adaptation from transcriptome data of juvenile R.canadum.In the normal,low and high salinity transcriptomes of R.canadum,110453,110510 and 110216 SNP sites were identified,with 60.72%,60.71%and 60.74%conversions,and 39.28%,39.29%and 39.26%transversions,respectively.All SNP sites of cobia are distributed on 28511 SNP-unigene.The functional annotation results show that there are 6480 SNP-unigene with GO annotation,13134 SNP-unigene with KOG annotation,and 5261 SNP-unigene with KEGG annotation.Further enrichment analysis revealed that 752 SNP-unigenes were annotated into multiple signal pathways related to salinity adaptation such as“Tight junction”.At the same time,346specific distributions of salinity adaptation-related candidate SNP sites were screened according to the distribution of SNP sites.Based on the standardized gene expression level analysis,26295 SNP sites were detected in the differentially expressed genes of R.canadum transcriptome,distributed among 7936 differentially expressed salinity-related genes.6.Cloning and expression analysis of AQP1 and AQP3 genes related to salinity adaptation of juvenile R.canadumThe cDNA sequences of AQP1 and AQP3 obtained by gene cloning technology are 741 bp and 744 bp,respectively,encoding 246 and 247 amino acids.The BLAST analysis results showed a high degree of similarity to Seriola dumerili,90.65%and92.71%,respectively.The results of q PCR showed that cobia AQP1 and AQP3 were expressed in 9 tissues,of which AQP1 was highest expressed in the kidney and least in the muscle,while AQP3 was highest expressed in the gill and least in the brain.After low-salinity adaptation,the expression of AQP1 gene was the highest in the kidney,followed by intestine,and the least gill;and after high salinity adaptation,the expression pattern of AQP1 gene was the same as that of low salinity adaptation.The AQP3 gene had the highest expression in the gills after low salinity adaptation,followed by the intestine and the least kidneys;after the high salinity adaptation,the AQP3 gene had the highest expression in the intestinal tissues,followed by the kidneys and the least expression in the gills.In summary,this study takes juvenile R.canadum as the experimental object,on the one hand,the effects of salinity changes on survival,growth,physiology,biochemistry and tissue structure of juvenile cobia were investigated.On the other hand,through comparative transcriptome analysis at the molecular level,preliminary studies were conducted on the mechanism of osmotic pressure regulation of juvenile cobia and the regulation of transcriptional regulation of genes related to salinity adaptation.At the same time,the genetic structure and expression levels of AQP1 and AQP3 genes of cobia were studied to explain the biological process and molecular regulation mechanism of cobia salinity adaptation,and provide a theoretical basis for the study of fish salinity adaptation. |
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