Study On Regulation Mechanism Of Lipid Metabolism In Scophthalmus Maximus Under High Temperature Stress

The adverse effects of high temperature stress on the growth performance of fish have been widely recognized,but there are few studies on the relationship between high temperature stress and energy metabolism,especially lipid metabolism.Lipid metabolism is an important and complex biochemical reaction in the body.As one of the three most important metabolisms,lipid metabolism can ensure the normal physiological function of the body and is of great significance to life activities.Lipids are important substances for energy storage and supply,as well as important structural components of biofilms.Abnormal lipid metabolism often leads to various diseases.In this paper,key genes and metabolites of lipid metabolism in turbot under high temperature stress were excavated for the first time through multi-omics analysis,and then the key metabolic pathways in lipid metabolism regulation were studied by means of molecular biology and cell biology,so as to analyze the regulation mechanism of lipid metabolism in turbot under high temperature stress.The results of this study will help to understand the environmental adaptation mechanism of turbot,promote the breeding of new varieties with temperature tolerance,have positive significance for the development of aquaculture,and open up a new research field for the regulation of energy metabolism.Especially lipid metabolism during temperature tolerance of fish.The main research contents and results are as follows:1.Integrated metabolome and transcriptome analysis revealed that heat stress affected lipid metabolism of juvenile turbotIn this experiment,turbot was cultured at different temperatures(20℃,25℃ and28℃),and kidney tissues were collected for transcriptome sequencing and metabolome sequencing,respectively.Differentially expressed genes and metabolites were identified based on transcriptome and metabolome data.Results showed that significant differences according to KEGG pathway analysis gene and metabolites were enriched in article 7associated with lipid metabolism pathways of steroid hormone biosynthesis,primary bile acid biosynthesis,glycerol phospholipid metabolism,linoleic acid metabolism,sheath lipid metabolism,and metabolic and unsaturated fatty acid glycerides biosynthesis.After correlation analysis of differentially expressed genes and metabolites in the pathway,The most representative 6 genes(lpcat2,Etnk1,TAZ,SCP2,ch25 hl and gpd1l)and 5metabolites(citicoline,UPD-6-sulfoquinoline,dihydroxyacetone,taurine and ophosphocholine)were selected according to the correlation coefficient.By analyzing the expression patterns of the selected genes and metabolites at different temperatures,it was found that they regulated lipid deposition and maintained lipid homeostasis in different degrees under heat stress,thus alleviating the disorder of lipid metabolism caused by heat stress.2.Transcriptome analysis was used to reveal the regulation pattern of lipid metabolism in juvenile turbot liver altered by high temperatureWhen analyzing the response of various tissues of turbot to high temperature stress,our team found that in addition to kidney,liver was also an important tissue responding to high temperature stress,and liver was also one of the important organs in the metabolic process of the body.Therefore,this experiment simulated the high temperature environment(20℃,24℃ and 28℃)that may occur during turbot cultivation.The response of liver lipid metabolism of juvenile Turbot under high temperature stress was studied by high-throughput sequencing technology.The results showed that 2067 differentially expressed genes(DEGs)in response to high temperature were identified by assembly and annotation of transcriptome data.After GO analysis,KEGG analysis and trend analysis,a total of 49 DEGs were found to be involved in lipid metabolism,which mainly focused on glycerophospholipid metabolism,steroid biosynthesis,glycerol metabolism,fatty acid metabolism and peroxisome proliferator-activated receptor(PPAR)signaling pathway.To further explore the possible transcriptional regulation of selected genes with significant differences in lipid metabolism,we constructed a regulatory network.We hypothesize that high temperature activates the PPAR signaling pathway through interactions with ligands such as fatty acids.On the one hand,HMGCS1 inhibits sterol synthesis by down-regulating the expression of key genes in the steroid biosynthesis pathway(SQLE,EBP and DHCR24).On the other hand,ACSL1 expression in this pathway was significantly increased at high temperature,which may play an important role in regulating fatty acid metabolism.In addition,we collected blood to detect changes in serum parameters,and found that triglyceride(TG),total cholesterol(TC)and low-density lipoprotein(LDL)levels showed a trend of first increasing and then gradually decreasing.These results indicate that liver lipid metabolism plays an important role in the regulation of high temperature stress in turbot,and the PPAR signaling pathway plays an important role in the regulation of lipid metabolism in response to high temperature stress.3.Cloning and characterization of PPARs gene in Turbot and its function in regulating lipid metabolism under high temperature stressPPAR signaling pathway is centered on PPARs family genes,which have been extensively studied for their important role in regulating lipid metabolism in mammals,but are rarely involved in Marine fish.In this study,PPARs family gene was cloned and subtypes of PPARs family gene were identified.Bioinformatics analysis of genes to understand gene structure and predict the possible role of genes.The expression of PPARs family genes was inhibited by inhibitor and RNA interference experiments,and the expression level of downstream lipid metabolism genes of PPARs was detected to elucidate the mechanism of PPARs regulation of lipid metabolism in turbot during heat stress.The results showed that there were three subtypes of PPARs gene in turbot,which were α,β and γ.The coding sequence(CDS)of PPARα is 1410 bp,encoding 469 amino acids.The CDS sequence of PPARβ is 1533 bp and encodes 510 amino acids.The CDS length of PPARγ gene was 1659 bp,encoding 552 amino acids.Structural analysis showed that all three subtypes had a ZNF-C4 domain and a HOLI domain.Phylogenetic analysis showed that the three isoforms were highly conserved during evolution.The m RNA level of PPARα was significantly increased under high temperature stress,and the expression of lipid synthesis genes FASN and SCD was significantly down-regulated.In vivo interference and cell inhibition of PPARα showed that the expression levels of FASN and SCD were significantly increased.q PCR results showed that high temperature activated the transcriptional activity of PPARβ and increased the activity of ACOX and CPT-1 genes.The expression of ACOX and CPT-1 was significantly inhibited when PPARβ was down-regulated by RNAi and inhibitor treatment at high temperature,suggesting that PPARβ is regulating the expression of ACOX and CPT-1 at high temperature,thereby regulating lipid catabolic activity.The expression level of PPARγwas significantly increased under high temperature stress,and the expression of fatty acid transporters FATP and CD36 were also significantly increased under high temperature.By interfering with PPARγ activity in vivo and inhibiting PPARγ activity in cells,the expressions of lipid transporters FATP and CD36 were significantly reduced.In conclusion,the three subtypes of PPARs are all activated by high temperature.After activation,the three subtypes play different regulatory roles to reduce lipid deposition by inhibiting lipid synthesis,promoting lipid catabolism and regulating lipid transport,respectively,so as to maintain lipid metabolism homeostasis in turbot.4.To investigate the effects of dietary taurine on growth performance and lipid metabolism of Turbot under high temperature stressIn the first part of experiments,taurine has been shown to play an important role in turbot response to high temperature.Taurine is an important nutrient.Taurine supplement in feed can promote the growth of the body and effectively relieve various stress reactions.However,the effect of taurine intake on lipid profile of turbot remains unclear.In this experiment,two temperature points were set up: normal temperature(15℃)and high temperature(24℃).Four diets with taurine levels(0%,0.4%,1.2% and 2%)were prepared and fed to turbot at each temperature to study the effects of taurine levels on growth performance of Turbot at different temperatures.The changes of lipid metabolism in turbot under high temperature stress and taurine addition were studied by combining molecular biology,pathology and lipidomic.The results showed that 0.4% ～ 2% taurine supplementation could promote the growth of turbot at 15℃,but only 2% taurine supplementation had positive effects on the growth and survival of turbot at 24℃.Histopathological findings showed that the morphology of renal units in the 15℃ group was normal and no obvious changes were observed.In the group without taurine at 24℃,the glomerular and interstitial capillary lumen were dilated and hyperemia was severe,the number of renal tubules was reduced,and some renal tubule epithelial cells were swollen and lumen was narrow.The glomerular swelling and interstitial congestion were significantly relieved by adding 2% taurine.Lipidomic combined with lipid index analysis showed that the contents of glycerophosphatides including PA,PE,PC and sphinolipids including Hex1 Cer,SM decreased significantly in response to high temperature stress,and increased significantly with the addition of taurine.The contents of glycerides such as TG and DG were significantly increased under heat stress,but remained high after taurine diet intervention.In conclusion,high temperature stress may affect the stability,permeability and fluidity of the biofilm by decreasing the content of phospholipid molecules in turbot kidney,and thus cause tissue damage.However,the addition of 2% taurine can alleviate tissue damage by increasing the content of phospholipid molecules.At the same time,high temperature stress can lead to lipid accumulation in turbot kidney tissue and induce dyslipidemia in vivo,but the addition of taurine cannot improve lipid accumulation.