| Sea urchin(Strongylocentrotus intermedius)is native to Japan and is now an important economic species in my country.It is widely cultivated in the Liaodong Peninsula and Shandong Peninsula.In recent years,global warming has intensified,hot weather in summer has occurred frequently,and large numbers of sea urchins have died in aquaculture.However,there are few reports on the molecular-level response and regulation mechanism of sea urchins intermedius in response to high temperature.To this end,this study used transcriptome and DNA methylation sequencing technologies to screen heat stress-related response genes and regulatory pathways,constructed a simplified genome methylation map,and identified the methylation modification patterns of genes under high temperature stress.The key candidate genes that DNA methylation regulates temperature,search for the correlation between gene methylation and gene expression in response to high temperature,in order to provide a certain theoretical basis for the cultivation of high temperature resistant varieties of sea urchin.The main research contents and conclusions are as follows:In order to find out the effect of temperature on the growth of sea urchins,based on the growth monitoring data of sea urchins with different shell diameters at different temperatures,using polynomial fitting and response surface method,a prediction model for the growth of sea urchins at different temperatures was established,and the results were obtained.Optimum and limit temperatures for growth of sea urchins with different shell diameters.The results showed that within the temperature range set in the experiment,the weight gain rate of sea urchins increased with the increase of temperature;when the temperature exceeded the optimum temperature,the weight gain rate of sea urchins decreased;when the water temperature exceeded the heat resistance limit of sea urchins,the sea urchins increased.Weight loss until death.The growth prediction model of the sea urchin affected by temperature is parabolic,and the R2 values of the models are all greater than 0.94,and the model construction is reasonable and effective.According to the model calculation,the optimal growth temperature of sea urchins with shell diameters of 2 cm,4cm and 6 cm are 16.1 ℃,15.4 ℃ and 13.6 ℃,respectively;the growth limit temperatures are25.1 ℃,24.5 ℃ and 21.0 ℃,respectively.1)In order to find out the effect of temperature on the growth of sea urchins,based on the growth monitoring data of sea urchins with different shell diameters at different temperatures,this paper uses polynomial fitting and response surface method to establish a prediction model for the growth of sea urchins at different temperatures,and find out the growth prediction models of sea urchins with different shell diameters.The optimum temperature and extreme temperature for the growth of sea urchins.The results showed that within the temperature range set in the experiment,the sea urchin weight gain rate increased with the increase of temperature;when the temperature exceeded the optimum temperature,the sea urchin weight gain rate decreased;when the water temperature exceeded the heat resistance limit of the sea urchin,the sea urchin lost weight.until death.The R2 values of the models are all greater than 0.94,and the model construction is reasonable and effective.According to the model calculation,the optimum growth temperatures for sea urchins with shell diameters of 2 cm,4 cm and 6 cm are 16.1℃,15.4℃ and 13.6℃,respectively;the growth limit temperatures are 25.1℃,24.5℃ and 21.0℃,respectively.2)Transcriptome sequencing of the two groups of sea urchins.A total of 41.68 G clean reads were obtained from 6 samples.The effective data volume of each sample was 6.49～7.07 G,the Q30 bases were distributed in 95.87～96.1%,and the average GC content was 42.65%,align the obtained clean reads to the reference genome,and the alignment rate is 99.9～99.92%.By comparison,the number of differential genes was 1968,of which 813 were up-regulated and 1155 were down-regulated.It was found that Amy,HK2,HSP70,CRYAB,PGK1,PKM,FDFT1,DHCR24,IDH1,Aco1,mdh1,RDH8,GGT1,CYP and other genes play an important role in the response of sea urchin to high temperature.Through GO and KEGG database annotation and enrichment analysis,GO enrichment analysis showed that the differential gene functions were mainly enriched in retinol metabolism,glutathione metabolism,and cholesterol synthesis.KEGG enrichment analysis of 328 metabolic pathways showed that the differential genes were mainly enriched in glycolysis,cytochrome P450 metabolism of foreign factors,steroid synthesis,glutathione metabolism and other signaling pathways.The genes related to high temperature response(Amy,HK2,HSP70,CRYAB,PGK1,PKM,FDFT1,DHCR24,IDH1,Aco1,mdh1,RDH8,GGT1,CYP)of sea urchin were preliminarily screened,and most of these genes were enriched in energy metabolism,immunity,Maintain cellular homeostasis and other processes.3)In this study,the Methyl RAD technique was used to measure the DNA methylation levels of high temperature-stressed sea urchin intestinal tissue and normal sea urchin intestinal tissue.The average number of CG methylation sites in normal sea urchin was 684,527,and the average number of CG methylation sites in high-temperature sea urchin was 680,788.High temperature did not cause a significant difference in the number of methylation sites in E.intermedius,nor did it change the methylation pattern of E.intermedius.The distribution of methylation sites on different functional elements of the genome shows that,except for the Splice Site Acceptor region,CWG sites are less than CG sites,and CG methylation sites are concentrated in the gene interval,CWG methylation sites Concentrate on gene and exon regions.There was a total of 12,129 CG differentially methylated sites between the two groups,of which 6,034 were up-regulated and6,095 were down-regulated,and there were 966 CWG differentially methylated sites,of which412 were up-regulated and 454 were down-regulated.GO enrichment analysis of differentially methylated sites showed that CG and CWG differentially methylated sites were mainly enriched in protein phosphorylation,membrane fusion,axon,and histone acetylation.KEGG enrichment analysis showed that endocytosis was regulated by PI3K-Akt signaling pathway,Erb B signaling pathway,AMPK signaling pathway,and actin cytoskeleton.The methylation type of sea urchin was mainly CG type,and high temperature did not change the methylation type of sea urchin.Most of the differentially methylated genes were significantly enriched in pathways such as energy and cell division,immunity,and nerve injury.It was speculated that the mechanism of sea urchin response to high temperature was involved in DNA methylation modification.4)Pairwise comparisons were made according to the expression or relative content data between m RNA and Methyl RAD,and the correlation between genes and Methyl RAD was calculated using the Pearson test,and the differentially expressed genes between Methyl RAD and m RNA were directly related to the gene name.Twenty-three genes were screened with differences in their methylation and gene expression,including PLXNA4,ft,Cox7a2,cah-3,Slc5a3,MKX,Orct,Moap1,pats1,HPSE,SULT1B1,bbs5,FAXDC2,Ids,GGT1,tll1,Kansl1,RBM34,SMCHD1,RDH8,PRADC1,Ighmbp2,Hint2.Ids and HPSE were simultaneously screened in both modes.Among them,15 genes were screened at the CG locus,including 8 positively correlated genes and7 negatively correlated genes.The negatively correlated genes were PLXNA4,ft,Cox7a2,MKX,pats1,HPSE,and FAXDC2.10 genes were screened at the CWG site,including 8 positively correlated genes and 2 negatively correlated genes.Negatively correlated genes include SMCHD1 and HPSE.PLXNA4 is mainly involved in axonogenesis,sympathetic nervous system development,signal transmission,immunity and other processes.Cox7a2 translates proteins to synthesize cytochrome c oxidase,and cytochrome c oxidase participates in the synthesis of oxidative phosphoric acid.MKX is mainly involved in the activity of DNA-binding transcription factors and the development of muscle organs.The expression product of HPSE is heparanase,which is mainly related to the inflammatory process of the body.SMCHD1 is mainly involved in epigenetic modification,gene silencing,DNA double-strand break repair and other functions.Candidate genes(PLXNA4,Cox7a2,MKX,HPSE,SMCHD1)driven by methylation in response to high temperature were initially screened. |
PDF Full Text Request