| Turbot（Scophthalmus maximus）,as a precious low-temperature marine fish imported from Europe,has extremely high commercial value.It has strict requirement on environmental indicators such as temperature.In particular,high temperature stress could cause a stress response in body,which lead to the increase of mortality,decrease of growth rate and disease resistance.Therefore,breeding of new high temperature tolerance varieties with excellent growth performance has become a top priority for healthy breeding[1].In the selection of fish resistance traits,the evaluation index of heat resistance is relatively single,which also cause problems such as an imperfect evaluation system for high temperature resistance selection,fewer molecular markers,low efficiency and so on.Therefore,research on molecular mechanisms of heat stress is of great significance for screening high-efficiency molecular（gene）markers.The heart is the first organ to form and affect during the embryonic development of vertebrates,and it plays a key role in maintaining body homeostasis[2].The function of the heart to cope with heat stress is an important factor for fish to set the upper thermal range in a high temperature environment.The study of the molecular mechanism of heart plasticity can solve the problem of ocean warming affecting the physiology and distribution of fish.This dissertation mainly explores the effects of turbot myocardial injury and cell apoptosis under heat stress,and uses second-generation sequencing technology to construct a turbot cardiac hyperthermic transcriptome database;based on the previous QTL localization of the research group and the analysis of multiple tissues transcriptomics studies,we have found that p53 plays a very critical role in turbot heat stress.At the same time,ubiquitin proteasome pathway which degrades redundant proteins in the body to maintain homeostasis was enriched.Exploring the regulatory relationship ofp53 and two DEG genes（ubiquitination-related genes mdmx,ube2h）will provide a data reference and theoretical basis for the study of the molecular mechanism of turbot heat stress.The specific research results are as follows:1.Turbot,as an economic fish adapted to cold water at low temperature,high temperature severely affects its growth and survival.The present study aimed to investigate in heart damage and mechanism the effects of thermal stress on physiologic,biochemical response andapoptosis gene expression multiple levels.The results showed that the aggravated degrees of swelling and breakage of myocardial fiber,dilatation of interstitial space,inflammatory cell infiltration,mitochondrial structure destruction and other tissue damagewith the elevated temperature,but the tissue damage was significantly reduced at 24℃-24h.CK activity increased significantly with the escalation of heat stress;LDH,SOD activity and MDA content reach their peak at 24℃.Expression levelsof Bax and Caspase-3 decreasing significantly after thermal stress,and the tendency were similar to them.However,the expression level of Bcl-2gradually increased.These results indicated that the myocardium could reduce the expression of Bax and Caspse-3 genes and promote the expression of the anti-apoptotic gene Bcl-2 to reduce the loss of myocardial cells to reduce thermal stress damage when it suffered a lesser degree of heat stress.Thissuggested that turbot suffered thermal stress,which lead the heart defense enzymes to exert resistance to maintains body homeostasis.The organism defense system itself is damagedbecause the heat stress exceeds its own physiological regulation threshold when the heat stress intensifies to28℃,which caused severely damages of heart structure and even lead to deathin turbot.The results showed that thermal stress could cause myocardial damage of turbot,and the body maintains homeostasis via regulating the activity of defense enzymes and apoptosis pathway related genes.This study provides a theoretical basis for subsequentresearch of the physiological adaptation mechanism of turbot and other fishes heart to thermal stress.At the same time,it provides more trait indicators for the high temperature tolerance traits to improve the breeding accuracy of marine fish.2.In order to explore the molecular mechanism of turbot resistance to high temperature and screen the genes related to high temperature resistance,this study used RNA-seq technology to perform transcriptome sequencing and bioinformatics analysis of turbot heart in four different temperature treatment groups（14℃,20℃,24℃,28℃）,including DEGs identification,GO gene function annotation,KEGG enrichment pathway analysis,qPCR verification of DEG genes,and mining of potential regulatory genes of the heart under heat stress.The main results obtained are as follow:611,375,686 original sequences and 592,822,038 valid sequences are generated,and the heat map analysis of hierarchical clustering is used to determine the distribution of DEGs in different groups.According to GO terminology,genes are distributed in more than 50 categories in biological processes,cellular components and molecular functions.3,214 genes include 891 genes in the biological process（BP）,1059 genes in the cell component（CC）,and 1264 genes in the molecular function category（MF）.Using KEGG database for pathway enrichment analysis,a total of 437,1128,2001,368,1071 and 683 genes were enriched in different pathways.After heat stress,a total of 34 pathways were significantly enriched（p<0.05）（p<0.05）（Figure 5）,including genetic information processes,metabolism,human diseases,and cellular processes.Among them,"protein processing in the endoplasmic reticulum（ER）"is the most enriched approach,which contains 70 genes.Other pathways include"DNA replication","lipid metabolism","amino acid metabolism","cell growth and death"and"cardiovascular disease".This study established a turbot cardiac high temperature transcriptome database,which provides abundant data reference for exploring turbot high temperature stress molecular mechanisms and screening genetic markers[3].3.Based on the previous QTL mapping and transcriptomics studies of multiple tissues,it was found that p53 plays a key role in turbot heat stress,and the ubiquitin proteasome pathway is enriched.Among them,mdmx and ube2h are the DEG genes.And studies have shown that the stability and activity of the p53 gene in the body are strictly regulated by ubiquitination.We conducted a preliminary study on the regulation of p53 via ube2h and mdmx gene in turbot under heat stress.（1）The research results of mdmx gene function are as follows:The results showed that the full-length cDNA sequence of Sm-mdmx gene was cloned by RT-PCR and RACE technology,and analyzed by bioinformatics.RT-PCR was used to detect the tissue expression of Sm-mdmx and the expression changes at four temperature points（14℃,20℃,24℃,28℃）in liver and heart tissues under heat stress.The transcription levels of Sm-mdmx and Sm-p53 in liver and heart tissues were detected by RNA interference technology at 6h,12h,and 18h after mdmx gene was knocked down under normal temperature and heat stress.The full-length cDNA sequence of turbot mdmx gene is 1623 bp.The 3’UTR region contains typical tailed signals AATAAA and poly A tail（Figure 1）.The ORF of the gene can encode a protein composed of 388 amino acid residues,with a relative molecular mass of 56.61k Da and a theoretical isoelectric point of 4.54.Turbot,Paralichthys olivaceus,and tilapia cluster together in evolutionary relationship,and have the closest genetic relationship with fish.Sm-mdmx mRNA level is highest in the spleen and lowest in the kidney.Sm-mdmx and p53 show antagonism effect under normal temperature conditions after mdmx interference,but show a synergistic effect in the heart under heat stress.It is speculated that there is an interaction between mdmx and p53 signaling pathways,and the interaction of mdm2 and mdmx also affect the expression changes of p53 gene,which need to be explored in the next step.（2）The research results of ube2h gene function are as follows:The results showed that the ube2h obtained by the previous team was detected by RT-PCR to detect the tissue expression of Sm-ube2h and the expression changes at the four temperature points in the liver and heart tissues（14℃,20℃,24℃,28）under heat stress.The transcription levels of Sm-ube2h and Sm-p53 in liver and heart tissues were detected by RNA interference technology at 6h,12h,and 18h after the gene ube2h was knocked down under normal temperature and heat stress.Sm-ube2h mRNA level was the highest in the spleen,higher in the heart,gills and brain,and lowest in the liver and kidneys.The expression of Sm-ube2h in the liver under heat stress increased with the prolongation of stress time,and the highest value appeared at 28℃.Although the level of Sm-ube2h in the heart also increased from 14℃,no significant difference was observed when the temperature reached 24℃ and 28℃.Sm-ube2h and p53showed antagonism effect under normal temperature after ube2h interference,but showed a synergistic effect under heat stress.It is speculated that there is an interaction between ubiquitin protease E2 and p53 signaling pathway.The results of the study showed that the p53 gene iis regulated by the ubiquitination-related genes ube2h and mdmx,and the expression of mdmx may have tissue differences in turbot.mdm2-mdmx-p53 worked together to maintain the homeostasis of body.In summary,this study explored the effects of turbot heart damage and cell apoptosis under heat stress,which determined the heat sensitivity of the heart.Then,the two key regulatory genes of p53,mdmx,ube2h,were screened through cardiac hyperthermia transcriptome sequencing.Cloning,characterization analysis and functional verification of gene were conducted,and it was preliminarily confirmed that the expression of turbot p53 gene is regulated by the negative regulator mdmx and ubiquitin-conjuagating enzyme ube2h.Ubiquitination could regulates the stability of Sm-p53 gene in response to turbot thermal stress,which has great significance.Our results improve our understanding of the molecular mechanism of heat tolerance in turbot and provide a theoretical basis for elucidating the molecular mechanism of Sm-p53 gene and ubiquitin proteasome pathway related genes response to heat stress in turbot. |
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