Characterization Of Physiological And Biochemical Response And Molecular Mechanism Of Thymallus Arcticus Grubei Under High Temperature Stress

Thymallus arcticus grubei is a famous economic fish unique to Heilongjiang Province,with gorgeous body color,tender flesh and delicious taste,which has high nutritional,ornamental and economic value.As a cold-water fish,its growth and survival require extremely strict environmental factors such as water temperature.However,under the global warming environment,continuous high temperatures and extreme weather occur frequently in the northern summer,and high temperatures have now become the primary environmental stressor limiting the healthy culture of Thymallus arcticus grubei.Therefore,three high temperature gradients(14℃,17℃ and 20℃)were set up in this study,with 11℃ as the control group,to carry out research on the physiological response and molecular regulatory mechanisms of Thymallus arcticus grubei under high temperature stress,and to provide basic data for the rational exploitation and resource conservation of Thymallus arcticus grubei.The main results are as follows:1.The results of serum biochemical indexes of Thymallus arcticus grubei under high temperature stress showed that the serum alanine aminotransferase(ALT)and glutamic oxalacetic aminotransferase(AST)activities in the 17℃ and 20℃ groups increased significantly(P < 0.05)with the increase of stress time;the serum alkaline phosphatase(ALP)activity and total protein(TP)content showed a decreasing trend(P < 0.05)with the increase of stress time;The differences in ALT,TP and ALP activities in the 14℃group at 48 h of stress were not significant(P > 0.05).The amylase and protease activities in the intestine of each high temperature group increased and then decreased with the prolongation of the stress time,while the lipase activity increased slowly.At 24 h of stress,amylase,protease and lipase activities in the intestine of the 17°C and 20°C groups increased significantly(P < 0.05);at 48 h of stress,intestinal amylase and protease activities decreased significantly(P < 0.05),while lipase activity increased significantly(P < 0.05).In addition,glutathione peroxidase(GPx)and superoxide dismutase(SOD)activities in the liver of the 17°C and 20°C groups increased significantly(P < 0.05)with increasing stress time,while catalase(CAT)activity decreased significantly(P < 0.05)at48 h of stress;antioxidant enzyme activities in the 14°C group decreased to the control level at 48 h of stress in both groups(P > 0.05).2.The results of the structural changes of liver of Thymallus arcticus grubei under high temperature stress showed that the liver tissue structure showed obvious pathological damage with the prolongation of stress time at 17°C.The liver tissue showed different degrees of nuclear atrophy and deformation and vacuolization after 12 h of stress,and the most serious damage of liver cells was observed at 48 h of stress,and even cell lysis occurred.The transcriptome sequencing results showed that a total of 214.44 G of valid data were obtained,with an average GC content of 49.45% and Q30 base distribution ranging from 93.48% to 96.06%.A total of 9144 differentially expressed genes(DEGs)were screened,and the number of DEGs showed a trend of increasing and then decreasing with the prolongation of stress,among which the maximum number of differentially expressed genes was 7148 at 24 h of stress.GO functional analysis showed that differentially expressed genes were mainly related to metabolism,catalysis and binding functions.KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in several energy metabolism and immune stress-related pathways,such as fructose and mannose metabolism,glycolysis/gluconeogenesis,protein processing in the endoplasmic reticulum,ribosome and PPAR signaling pathways,etc.Among them,mdh,gapdh and grhpr genes were significantly down-regulated in energy metabolism pathway(P < 0.05);immune stress The genes of hsp 70,hsp 90,ubc and erk were significantly up-regulated(P < 0.05)3.Obtaining the full-length c DNA sequence of the heat shock protein 90 gene(TA hsp90)of Thymallus arcticus grubei.The results showed that the full-length c DNA sequence of TA hsp90 gene was 2794 bp,containing 5’ UTR length 273 bp,3’ UTR length346 bp,open reading frame length 2175 bp(ORF),encoding 724 amino acids.TA Hsp90 amino acid sequence had five characteristic sequence tags of heat shock protein 90 family and a conserved MEEVD structure at the C-terminus.The predicted relative molecular mass of TA Hsp90 protein is 83.31 KDa with an isoelectric point value of 4.99;there is no signal peptide or transmembrane region;the protein spatial structure is mainly composed of α-helix with irregular coiling.The homology and evolutionary tree analysis showed that the amino acid sequences encoded by TA hsp90 gene were more than 91%homologous with salmonids,with the highest homology(99.59%)with herring-like whitefish.The tissue distribution results showed that TA hsp90 gene was expressed in nine tissues with tissue specificity,and was highly expressed in heart,muscle and liver tissues.Under high temperature stress,the expression of TA hsp90 gene in liver tissues showed a trend of increasing and then decreasing with the prolongation of stress time,and reached the maximum value at 24 h of stress(P < 0.05);among them,the expression of TA hsp90 gene in 17℃ and 20℃ groups decreased significantly at 48 h of stress(P <0.05).In this study,we initially constructed a heat stress model for Thymallus arcticus grubei,and investigated the effects of high temperature stress on serum physiological and biochemical responses,liver tissue damage and differential gene expression of Thymallus arcticus grubei,and conducted the cloning and identification of heat stress-related genes,the results of which can lay a good scientific foundation and theoretical basis for the response of Thymallus arcticus grubei to high temperature stress and molecular regulatory mechanisms.