Study On The Physiological Response And Molecular Mechanism Of Takifugu Fasciatus To Low Temperature Stress

In the present study,high-throughput sequencing methods such as transcriptomics,proteomics and metabolomics were carried out to study the response mechanism of T.fasciatus under low temperature stress.Then,we further explored the effects of salinity on liver function,oxidative stress,lipid metabolism,p38 MAPK,immunity apoptosis and survival of T.fasciatus under low-temperature.This research means to systematically explore the molecular regulatory mechanism of T.fasciatus in response to low-temperature stress,and to provide theoretical basis for the overwintering of anadromous fishes.The results of this study mainly include the following two parts:1.Integrated application of multi-omics provides insights into cold stress responses in pufferfish T.fasciatusAt transcriptional level,a total of 5166 differentially expressed genes were identified,including 2544 up-regulated and 2622 down-regulated genes.GO enrichment analysis of these differential genes showed that the genes regulated by T.fasciatus in response to low-temperature stress were mainly clustered in biological GO clusters such as binding,ion binding,intracellular,intracellular part,cellular response to DNA damage stimulus,macromolecule catabolic process and so on.In addition,KEGG analysis showed that some important pathways in liver were involved in the regulation of low-temperature stress,such as ibosome biogenesis in eukaryotes,glyoxylate and dicarboxylate metabolism,RNA transport,PPAR signaling pathway and fatty acid biosynthesis.At the level of translation,we conducted quantitative proteomics on T.fasciatus liver using iTRAQ.This is the first time that iTRAQ has been applied to study cold tolerance in fish.A total of 3741 proteins were identified,and 160 were differentially expressed,including 53 up-regulated and 107 down-regulated proteins.Subsequently,11 genes such as HSP90,CIRB,GST,RAP1 A,ERBB2,FLNB,RPS6 KA,DAAO,COX5 A,A2ML1 and CAB 39 were verified by qRT-PCR.PRM verified the abundance of HSP90,CIRB,GST,FLNB and A2ML1 proteins,and confirmed the reliability and repeatability of our data.Two signal transduction pathways,MAPK and Wnt,were enriched by KEGG.Based on the results,most of the differentially expressed proteins were noted in those involved in oxidative stress(9 proteins,HSP90,CIRP,CSDE1,DAAO,GST,ZIP7,RDH12,TFRC1,QSOX1),mitochondrial enzymes related to transport(11 proteins,PRODH,TOMM20,OAT,Ucp1,C8 G,COX5A,ATP5 J,GATA,MPC1,PNPT1,THRS)and signal transduction(13 proteins: RAP1 A,ERBB2,FLNB,RAC1,RPS6 KA,CAB39,CSNK1 A,IMPA1,MTM14,PIK3,G protein,PLCB,CACYBP).At metabolic level,an untargeted metabolomics analysis was performed in livers using a UHPLC-Q-TOF MS platform.The experiments of QC sample spectrum comparison,principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA)show that the instrumental analysis system in this experiment was stable and reliable.A total of 4,085 positive ion peaks and 5,379 negative ion peaks were identified.In the positive ion mode,a total of 40 differential metabolites were identified,including 9 down-regulated and 31 up-regulated metabolites.Integrative analysis of regulatory pathways involved in differential metabolites,the number of pathways involved in lipid metabolism is the largest,which indicated that lipids were the key to cope with low-temperature stress.Combined transcription-protein-metabolism analysis revealed that the lowtemperature tolerance mechanism of T.fasciatus focused on 20 pathways,including 14 up-regulated and 3 down-regulated differential metabolites,8 up-regulated and 3 downregulated genes,and 3 reverse-trend genes.The interaction between them was clustered mainly into two clusters.One of these was related mainly to the transmembrane transport of bile salts,and the other to unsaturated fatty acids,vitamins and adenosine.These results suggested that the regulation of low-temperature tolerance was mainly achieved by enhancing the metabolism of unsaturated fatty acids,bile salt transport,vitamin uptake and antioxidant capacity.In addition,a total of 23 potential SNPs were detected in the 11 differentially expressed genes with the same trend.Subsequently,we extracted the DNA of 50 individuals and verified the SNPs.A total of 13 SNP were positive.These SNPs will be further confirmed in the follow-up work to determine whether they can be used as molecular markers to distinguish the cold-tolerant T.fasciatus.2.Effects of salinity on physiology and biochemistry of T.fasciatus at lowtemperatureIn the study of low-temperature tolerant mechanism,we found that lowtemperature can affect the expression of signal transduction,oxidative stress,mitochondrial enzymes and other related genes and proteins,and then hinder the normal metabolism of liver,changing the levels of oxidative stress,lipid metabolism and immune system.Adding appropriate salinity in water environment can effectively alleviate the pressure caused by low-temperature.The results are as follows:(1)reduce oxidative stress level.After the addition of 10 ppt salinity at 13 and 17℃,the concentration of GSH-PX,cortisol and MDA,expression of HSP90 were decreased,comparing to those in control fish.(2)Alleviating lipid metabolism.The stimulation of 10 ppt salinity could significantly increase T-CHO at 13 and 17 ℃.On the contrary,GLU and TG showed an upward trend,and the content of GLU and TG decreased when 10 ppt salt was added at 13 or 17 ℃.(3)To altering immune system.The activities of ALT,AST,IgM and LZM,the number of Gran,RBC and LYMPH and the expression of IFN,IFNR,IL-4 and IL-4R have changed significantly.(4)Altering the expression of p38 MAPK and its phosphorylated protein,and the expression of downstream transcription factor-related genes(ATF2,ElK-1,MEF2 and P53)of p38 MAPK.The changes of these genes and proteins correspondingly changed the survival rate and apoptotic index of liver in different treatments.At 13 and 17 ℃,the mortality time of fish treated with 10 ppt was relatively delayed,comparing to that of 0 and 20 ppt.The mortality rate of 10 ppt salinity treatments was significantly lower than that of 0 salinity treatments at 13 ℃,and was more significant with the increase as the time pass.Similarly,at salinity of 0 and 10 ppt,the apoptotic level increased with the decrease of temperature.Moreover,10 ppt of salinity at 13 and 17 ℃ could significantly reduce the apoptotic level compared with 0 and 20 ppt of salinity.No significant change in apoptotic level was observed at 21 ℃,but high apoptotic level was observed at 2025(20 ppt,25 ℃).These results suggested that there was a mechanism between salt and low-temperature to regulate the physiological steady state of T.fasciatus.