Regulation Of Energy Metabolism Of Apostichopus Japonicus Via Methylation Modification In Response To Heat And Hypoxia Stress

Aquatic organisms will be stressed by different factors in their living environment,including abiotic factors and biological factors.Heat stress is the most common abiotic stress,which has a series of serious effects on the physiological and metabolic behavior of organisms,and even cause individual death.At the same time,the decrease of dissolved oxygen in heat water can also cause hypoxia stress to organisms,and further aggravate the burden and damage of tissues.At present,studies have found that energy metabolism regulation is one of the important regulatory mechanisms for organisms to resist heat and hypoxia stress,including the production of ATP and glycolytic intermediates.The imbalance of ATP supply and demand in organisms under heat and hypoxia stress can use acclimation to reshape energy homeostasis,and then form a heat acclimation phenomenon to adapt to heat and hypoxia stress.The regulation mechanism is realized by methylation modification to change chromatin structure and transcription and translation of heat and hypoxia response genes,which is of more significance in aquatic organisms more vulnerable to heat and hypoxia stress.In this paper,five indexes indicating the changes of energy and metabolism: ATP content,ATPase activity,pyruvate kinase activity,mitochondrial content and mitochondrial membrane potential were selected to detect the changes of energy metabolism after Apostichopus japonicus responding to heat and hypoxia stress.Then,the whole genome bisulfate sequencing(WGBS)technology was used to analyze the DNA methylation of the respiratory tree of A.japonicus under heat,hypoxia and heat-hypoxia stress,screen the circadian rhythm pathway and calcium signal pathway,and further explore the relationship between key genes in the pathway and energy metabolism.Protein arginine methyltransferases(PRMTs)were screened based on proteomics to analyze the relationship between protein methylation and energy metabolism.This study is expected to clarify the mechanism of A.japonicus in response to heat,hypoxia and heat-hypoxia stress from DNA level and protein level methylation.The main results are as follows:(1)The related indexes of energy metabolism of A.japonicus under heat,hypoxia and heat-hypoxia stress were measured.The ATP content,ATPase activity,pyruvate kinase activity,mitochondrial content and mitochondrial membrane potential of A.japonicus were measured after continuous stress of heat(26 ℃,DO 5 mg/L),hypoxia(16 ℃,DO 2 mg/L)and heat-hypoxia(26 ℃,DO 2 mg/L)for 6 h,12 h,24 h,48 h,5 d and 7 d.ATP content,ATPase activity and pyruvate kinase activity all increased first and then decreased.The activity of pyruvate kinase decreased to the lowest and the mitochondrial membrane potential decreased at 12 h of heat,hypoxia and heat-hypoxia stress;After 48 hours of stress,pyruvate kinase activity reached the peak and mitochondrial membrane potential also increased.The results showed that A.japonicus could meet the energy demand through the dynamic regulation of aerobic metabolism under heat,hypoxia and heat-hypoxia stress.The microscopic observation results of the respiratory tree tissue of A.japonicus after heat,hypoxia and heat-hypoxia stress showed that the connective tissue shrank significantly,and the dendritic epithelial tissue dispersed and arranged irregularly.This result showed that the respiratory tree was an important organ for A.japonicus to respond to heat and hypoxia stress and respond quickly.(2)In order to further clarify the regulatory pathway of energy metabolism transformation,the whole gene methylomics of A.japonicus respiratory tree under heat,hypoxia and heat-hypoxia stress for 48 hours were analyzed by WGBS technology.The results showed that 4% of genomic C sites of A.japonicus were methylated,and the main forms of methylation were CG,CHG and CHH(where H was a,C or T),and the proportion of CG sites was as high as 25.522%,which was much higher than that of CHG(0.585%)and CHH(0.597%).After comparing the differentially methylated regions(DMRs)with the whole genome,it is divided into promoter associated DMRs and gene associated DMRs.Go analysis showed that DMRs in the promoter associated DMRs and gene associated DMRs were mainly enriched in cellular processes,metabolic processes,cell membrane components and catalytic activities.KEGG analysis showed that gene associated DMRs significantly enriched endocrine resistance,Th1 and Th2 cell differentiation and thyroid hormone signal transduction pathway,and the promoter associated DMRs significantly enriched circadian rhythm pathway,calcium signal pathway,oxytocin signal pathway and apelin signal pathway.(3)In order to clarify the pathway of methylation regulating circadian rhythm key genes mediated by heat and hypoxia stress,we screened five rhythm genes from A.japonicus genome and whole gene methylomics.Cryptochrome-1a(CRY1a),cryptochrome-1b(CRY1b),aromatic hydrocarbon receptor nuclear translocator like 1(BMAL1),clock gene(CLC)and timeless gene(TIM)were cloned,and CRY1 a promoter region(site 299378-site 299529)and CRY1 b promoter region(site 52083-site 52112),CLC promoter region(site 3142-site 3218),BMAL1 exon 1 region(site 280973-site281011)were obtained.The Cp G island of CRY1 a promoter could be methylated under heat,hypoxia and heat-hypoxia stress,and the methylation probability was significantly increased by 30% under heat stress;Under the same conditions,only site 84 on the Cp G island of CRY1 b promoter significantly increased by 30%.Double luciferase assay showed that the methylation of Cp G island in CRY1 a promoter and the methylation of Cp G island site 84 in CRY1 b promoter inhibited the promoter activity of Cp G island.The results showed that the expressions of CRY1 a,CRY1b and BMAL1 were significantly up-regulated after heat stress,and the expressions of CRY1 a,CRY1b,BMAL1,CLC and TIM were down-regulated after hypoxia and heat-hypoxia stress.After RNA interference of CRY1 a and CRY1 b,pyruvate kinase and ATPase activities were significantly up-regulated,while mitochondrial membrane potential was significantly down regulated.The results showed that A.japonicus affected the expression of CRY1 a and CRY1 b through methylation modification of CpG island in promoters,and further regulated energy metabolism in response to heat,hypoxia and heat-hypoxia stress.(4)In order to determine whether the change of calcium regulated by methylation responds to heat and hypoxia stress,we screened rynodine receptor(RYR)in calcium signaling pathway from methylomics,and amplified two Cp G island sequences(RYR heat Cp G island and RYR hypoxia Cp G island)from methylomics under heat stress and hypoxia stress.The results of methylation sites showed that the methylation probability of RYR hypoxia Cp G island decreased by about 20% under hypoxia stress and heat-hypoxia stress;Under the same conditions,the methylation level of RYR heat Cp G island did not change significantly.The effects of heat,hypoxia and heat-hypoxia stress on the expression of RYR in cavity cells and respiratory tree of A.japonicus were further detected.RYR was significantly up-regulated in cavity cells and decreased in respiratory tree under hypoxia and heat-hypoxia stress.In order to further explore the regulatory mechanism between RYR and calcium,the changes of calcium in A.japonicus under heat,hypoxia and heat-hypoxia stress for 6 h,12 h,24 h,48 h,5 d and 7 d were detected.Within 12 hours of hypoxia and heathypoxia stress,the calcium in A.japonicus showed a significant downward trend.After RNA interference with individual A.japonicus by ds RNA RYR,the calcium content in increased significantly,and the mitochondrial content,mitochondrial membrane potential and ATP content of interfering individuals were significantly up-regulated after hypoxia and heat-hypoxia stress.This shows that methylation regulates RYR expression,affects the changes of calcium,and then regulates the changes of energy metabolism in response to hypoxia and heat-hypoxia stress.The results showed that the calcium concentration was rhythmic,but not affected by external stress conditions.Further interfering with CRY1 a and CRY1 b,it was found that calcium was only regulated by CRY1 b.This study shows that calcium not only have rhythm,but also are affected by methylation modified RYR,and RYR can promote the increase of calcium content,ATP content and related energy metabolism indexes under hypoxia and heat-hypoxia stress.(5)Based on proteomics,the mechanism of energy change mediated by protein methylation in response to heat and hypoxia stress was studied.Three Putative methyltransferase DDB＿G0268948 with the most significant expression were screened from the proteome of A.japonicus under heat,hypoxia and heat-hypoxia stress.The three proteins contain two conserved regions: AA(V,I)DV(I)GCGT(S)G and V(T)LL(K)P(H)G(Q)GCF(V)A(L)Q(V),which constitute the N-terminal and C-terminal of METTL11 domain.Multiple sequence analysis showed that the two conserved regions were highly conserved with arginine protein methyltransferase,which was preliminarily identified as arginine protein methyltransferase(PRMTXIs).The open reading frame of PRMTXIs was amplified,and the recombinant vector with green fluorescent protein was constructed and transfected into carp epithelial cells.The cell localization experiment showed that PRMTXI-1 protein was localized in the nucleus,while PRMTXI-2 and PRMTXI-3 were localized in the cytoplasmic matrix.The relative expression of PRMTXI-2 and PRMTXI-3 under heat and hypoxia stress is consistent,which is different from that of PRMTXI-1.This shows that PRMTXI-1 may play different regulatory roles from PRMTXI-2 and PRMTXI-3.After PRMTXIs recombinant vectors were transfected into primary cavity cells of A.japonicus,the ATPase activity,mitochondrial membrane potential and mitochondrial content decreased significantly.The results showed that A.japonicus could regulate the energy metabolism pathway by up regulating the expression of PRMTXIs,and then respond to heat,hypoxia and heat-hypoxia stress.