Revealing Osmotic Pressure Regulating Mechanism Of Takifugu Rubripes Based On Transcriptome Technology

Currently,seawater fish desalination or low salinization is one of research hotspots.Takifugu rubripes has a high economic value.With its aquaculture industry is growing,there are a variety of factors restrict the breeding process.Low salt or desalination domestication can achieve the possibility of inland breeding of marine fish and reduce the limitations of breeding.The aim of this study was to clarify the osmotic pressure regulation mechanism of T.rubripes,especially at the cellular and molecular levels.Due to explore the mechanism of osmotic pressure regulation,histological and molecular biology methods,as well as the expression of genes,were studied under different salinity.In this study,the transcriptional level of genes and tissue structure of T.rubripes under sudden and gradual change of salinity were studied.Both groups of salinity 32 and salinity 4 were set in the gradient salinity experiment for 30-day.Each group had three parallels.After breeding,gills were sampled for transcriptome sequencing.Gills,kidneys and intestine were fixed for tissue slices.The control group(salinity 32),salinity 16,salinity 12,salinity 8 and salinity 4 five salinity groups were set in the sudden salinity experiment.Gills and kidney were sampled at 3 h,6 h,12 h,24 h,48 h and 72 h to determine gene expression.A total of 378 genes were identified after transcriptome sequencing.Among them,176 genes were up-regulated and 202 genes were down-regulated.Ten genes were randomly selected for real-time quantitative PCR(qRT-PCR).The results of qRT-PCR were in consistent with the sequencing results.In order to further explore the effect of salinity on the gene expression level of the T.rubripes larvae,two genes,heat shock protein 70(Hsp70)and(Immunoglobulin M,IgM)related highly to salinity were selected in this study.Three genes,cytochrome P450 1A1(CYP1A1)gene,the solvate carrier family 39A6(SLC39A6)gene,Immunoglobulin M(IgM)and glucose-6-phosphate isomerase(GPI)gene were used to study the gene expression of T.rubripes under salinity change suddenly.The results showed that reduction of salinity had an effect on the five genes,but the degree of influence was different.The expression of Hsp70 gene in gill increased more strongly than that in kidney.In kidney,the expression level of Hsp70 gene was significantly higher than that of control group at the same time point after 12 h(P <0.05).At 12 h,at the same time(P <0.05),the expression level of IgM gene in the low salinity group was significantly higher than that in the control group(P <0.05).The expression of CYP1A1 in the gills was higher than that in the control group at the same time point(P <0.05).The expression of CYP1A1 in the kidney was different from that in the control group at salinity 4(P <0.05).Compared with the control group,the expression of SLC39A6 gene in the low salinity group was significantly higher than that in the control group at 3h(P <0.05).In the kidney,at the same time(P <0.05),the expression level of GPI gene in low salinity group was significantly higher than that in control group at 3 hours under low salinity treatment(P <0.05).Structural changes in gill,kidney and intestine of juvenile T.rubripes with body weight of(11.34±2.13)g were observed under low salinity treatment by using the histological photomicrography technique.The relationships between structural changes and osmotic regulation were discussed.The results showed that the widths of gill filaments and gill lamellae significantly increased and the space of adjacent gill filaments reduced significantly under low salinity treatment(P<0.05).The gill lamellae cells became more plumps.The number of chloride cells were(0.67±0.82)cells/100?m,which was obviously lower than the control group(P<0.05).Compared with the control group,the glomerular expanded and the gap between the glomerular and the inner wall of the renal capsule became significantly smaller.In the low salinity group,thickness of simple columnar epithelium was(51.33±2.27)?m,remarkablely higher than the control group(P<0.05).The number of goblet cells((5.20±0.79)cells/100?m)among columnar epithelial cells decreased significantly(P<0.05)while the body size of goblet cell had no significant difference within two groups(P>0.05).When the salinity changes,the gene expression level and tissue structure of juvenile T.rubripes changed,which is beneficial to enhance the environmental adaptability of T.rubripes.This study can provide the basic biology materials in the field of histology and molecular studies for the low salinization of T.rubripes.And this study can also provide a theoretical basis for the further study of the osmotic adjustment mechanism of T.rubripes.