| Scophthalmus maximus is a unique and valuable low-temperature economic fish native to the European coast.High temperature resistance is an important trait of turbot and a major target of genetic improvemen,and molecular markers and functional genes closely linked to high temperature traits through QTL mapping are important tools for molecular genetic breeding.Based on the rich genetic population,we completed the construction of high-density genetic linkage map.In this paper,QTL mapping for high temperature tolerance of turbot was studied.Genome-wide comparison and annotation were performed on the locational regions to screen candidate genes related to temperature tolerance.The effect of related genes in the enriched P53 signaling pathway in the QTL interval under high temperature stress was preliminarily explored.This study provides a theoretical basis for the breeding and molecular mechanism of turbot resistant to high temperature.The details are as follows:Based on the high-density genetic linkage map,this study carried out QTL mapping and candidate gene screening for high-temperature resistance traits.The results showed that a total of seven QLTs were identified,which were linked to high temperature resistance traits,which were located in LG17,LG17,LG17,LG20,LG21,LG4 and LG6.Among these QTL linkage groups,LG17 had the highest LOD(5.77),explaining 36.3% of the phenotypic variation,whereas LG17(81.113 cM)had the lowest LOD(3.08),explaining 21.4% of the phenotypic variation.The marker distance of high temperature resistant QTL interval ranged from 1.75 cM to 25.07 cM,and contained a total of 88 SNPs and 2 SSRs.We obtained 535 genes for genetic annotation of seven high temperature QTL intervals using bioinformatics tools.Using KEGG enrichment and GO functional classification analysis showed that metabolic regulation,signal transduction,and cellular process pathways accounted for a considerable proportion.It indicates that the physiological and biochemical self-balancing process occurs during the high temperature stress of turbot.The results of this study also show that the QTL mapping has reliable scientific significance for functional gene mining and analysis.In this chapter,we exerted acute high temperature stress on turbot to identify their response in the expression of four candidate genes including UBE2 H,p53,ZNF469,and MAGI2 genes in the Liver,gill,spleen and skin.These genes are derived from the QTL related to high temperature resistance which located at high-density genetic linkage maps.Relative mRNA expression of UBE2 H,p53,ZNF469,and MAGI2 in five temperature groups(20?,23?,25?,28?)and a control group(14?)were tested using quantitative real-time PCR.Data analysis demonstrated that the four genes expressed in all tissues in a tissues and temperatures specific pattern.The relative mRNA expression of UBE2 H firstly increased and then decreased in all four tissues.The expression levels in the liver,spleen,and skin tissues increased at rapidly 20? and reached a peak value(P<0.05),and peaked at 23? in gill tissue(P<0.05).The expression of P53 mRNA increased first and then decreased in all four tissues.However,the expression of p53 increased sharply and reached a peak at 28? which was significant(P<0.05).It was speculated that the tissue was severely damaged and the cells are heavily apoptotic.ZNF469 and MAGI2 were abundantly expressed at 20? in all four tissues with over than other temperatures.Therefore,we thought that both can serve as biomarkers for response to high temperature stress in turbot.In order to further understand the role of p53 gene of Scophthalmus maximus in stress response under high temperature stress,the full-length cDNA of Sm-p53 gene was amplified by RT-PCR and RACE,and bioinformatics analysis was performed.Real-time quantitative PCR was used to detect the expression of Sm-p53 mRNA and its expression characteristics at five temperature points(14?,20?,23?,25?,28?)during chronic warming,and detected by Western Blot.Expression characteristics of SM-P53 protein in chronic warming experiments.The results showed that the full-length cDNA of Sm-p53 gene was 2928 bp,open reading frame was 1146 bp,encoding 381 amino acids,the isoelectric point of the encoded protein was 6.73,and the molecular weight was 42.77 kDa.The Sm-p53-encoded amino acid sequence has a typical DNA-binding domain and a tetramerization domain.The P53 protein sequence contains a DNA-binding domain and a tetramerization domain.Homology analysis of the amino acid sequence encoding Sm-p53 indicated that the fish p53 gene was clustered into one,and the turbot was closely related to the gingiva,with zebrafish.Real-time fluorescence quantitative detection showed that the Sm-p53 gene was expressed most in the liver,followed by sputum,which was significantly higher than that of muscle tissue.(p<0.05);In the chronic warming experiment,the expression level of Smp53 gene generally appeared to rise first and then decrease with increasing temperature.The results of Western Blot showed that the expression of SM-P53 protein increased first and then decreased with the increase of temperature during the chronic warming,and the expression level was the highest at 23 ?,and the expression at 14 ? was the lowest in the control group.The results showed that Sm-p53 gene is closely related to the heat stress response of turbot,and plays an important role in the body's anti-reverse response.It provides a theoretical basis for elucidating the molecular mechanism of Sm-p53 gene involved in the body's stress response in turbot.The above experimental results indicate that Sm-p53 gene plays an important role in the response to heat stress in turbot.Summary,this study screened key genes related to temperature resistance traits by QTL mapping in the high temperature genetic linkage map of turbot,and cloned and analyzed one of the key genes Sm-p53 gene.Functional Verification.In order to reveal the molecular mechanism of fish stress response under high temperature stress,it lays a foundation for further guiding the breeding of high temperature resistant families. |
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