| Sebastiscus marmoratus is a kind of ovoviviparous fish widely distributed in the coastal areas of China,Japan,Korea and the Philippines.It has certain economic value and is one of the ideal fish species for offshore aquaculture.It often inhabits in the nearshore rock bottom environment,and its optimal temperature and salinity will change with the change of the growth stage.Temperature and salinity are important physical and chemical factors in aquatic environment,which are closely related to the growth,development and survival of fish.Temperature stress directly affects the physiological activities of fish feeding,metabolism and immunity.Salinity,on the other hand,affects physiological function through osmotic pressure changes.At present,due to the influence of global climate,extreme weather(typhoons,rainstorms,etc.)occur frequently,leading to drastic changes in water temperature and salinity in a short period of time.Transcriptome can be used to study gene expression,gene structure and gene function in specific tissues and environments,and elucidate the molecular mechanism of species response to environmental stress.On the basis of mRNA studies,whole transcriptome covers a variety of non-codingRNAs and the analysis of their regulatory networks,which can further explore the internal regulatory mechanisms among variousRNAs.To assess the impact of environmental factors on S.marmoratus,the whole transcriptome was sequenced from the liver of the S.marmoratus under acute temperature stress,and the transcriptome was sequenced from the gill of the S.marmoratus under acute salinity stress in this study.We have unearthed the key genes and pathways involved in temperature and salinity regulation;and the long non-codingRNA(lncRNA),microRNA(miRNA)and mRNA of temperature adaptation of the S.marmoratus were analyzed,the lncRNA-miRNA-mRNA regulatory network was constructed to further understand the internal regulation mechanism of the non-codingRNA network;the genome-wide identification of heat shock protein 70(HSP70),an important functional gene family,was conducted to explore the temperature regulation mechanism of the HSP70 gene family.This study laid the foundation for elucidating the mechanism of the response to environmental stress S.marmoratus at the molecular level.It mainly includes the following aspects:1.Whole transcriptome analysis of the liver of S.marmoratus under acute temperature stressWith 20°C as the control group,the S.marmoratus was subjected to acute temperature stress at 15°C and 25°C.After 12 hours,the liver tissue was taken for whole transcriptome sequencing,and the sequencing results of mRNA,lncRNA and miRNA were analyzed.(1)mRNA related results: compared with the control group,376 differentially expressed genes(DEGs)were found in the high-temperature group;59 DEGs were found in the lowtemperature group,and 740 DEGs were found between the high-temperature group and the low-temperature group.These DEGs functions were mainly concentrated in GO entries such as metabolic process,molecular function,biological process,catalytic activity,mitochondrial inner membrane,and binding.Compared with the control group and the lowtemperature group,the DEGs in the high-temperature group were significantly enriched in protein processing in endoplasmic reticulum pathways.Compared with the control group,the DEGs in the low-temperature group were significantly enriched in the fatty acid degradation and amino sugar and nucleotide sugar metabolism pathways.This may be because high-temperature stress can cause heat shock proteins to be stimulated to participate in stress regulation to prevent protein misfolding and translocation,and reduce body damage;the glycolysis rate will decrease under low-temperature stress,amino sugar and nucleotide sugar metabolism will be changed to regulate,and fatty acid degradation can provide energy for low-temperature stress.(2)lncRNA related results: compared with the control group,147 differential lncRNAs were found in the high-temperature group;59 differential lncRNAs were found in the low-temperature group;and 189 differential lncRNAs were found between the high-temperature group and the low-temperature group.The number of differentially expressed lncRNAs in the low-temperature group was significantly less than that in the hightemperature group.To a certain extent,the stress of low-temperature on S.marmoratus may be less than that of the high-temperature group.(3)miRNA related results: a total of 233 known miRNA mature and 469 novel miRNA mature were obtained.The results of differential expression analysis showed that there were a total of 40 miRNAs differentially expressed in the high-temperature group compared with the control group;and 39 miRNAs differentially expressed in the high-temperature group with the low-temperature group;the low-temperature group was compared with the control group,and a total of 44 differentially expressed miRNAs were obtained.There were no shared differentially expressed miRNAs in each combination,and the unique miRNAs of each combination may specifically respond to the temperature stress of S.marmoratus,which can be further analyzed in future research.(4)The correlation analysis of miRNAs and its target lncRNAs and target genes found that compared with the control group,there were two key miRNAs(miR-205-5p and miR-456)in the high-temperature group,which positively regulate FKBP prolyl isomerase 4、heat shock protein 4a and aprataxin expression,so as to avoid protein folding and DNA damage caused by high temperature,and effectively reduce the damage of the body under high temperature;in the low-temperature group,only one miRNA was included in the network,and the whole regulatory network was simpler than that in the high-temperature group,indicating that the effect of low temperature on S.marmoratus may be less than that of high temperature,and it does not need to mobilize too muchRNAs to maintain body stability.2.Expression analysis of HSP70 in S.marmoratus under acute temperature stressWe conducted a genome-wide identification of the HSP70 gene family of S.marmoratus,identified and named 15 HSP70 family genes.Phylogenetic analysis using HSP70 family genes of different species found the following points:(1)All HSP70 family genes in S.marmoratus were well clustered with HSP70 family genes of other fishes,indicating the genetic relationship between hsp70 genes corresponding to different species was closer than the genetic relationship between different hsp70 genes of the same species.(2)Except for zebrafish,the hsph1 gene didn’t appear in S.marmoratus or other teleosts.However,it is not yet certain whether hsph1 really disappeared from the S.marmoratus genome.(3)Teleosts have more gene duplications than mammals,which may be related to the fact that teleosts have experienced whole-genome duplication.(4)Compared with Tetrapods,more paralogues of hspa8 and hspa4 were found in teleosts.The teleost-specific genes indicated that hspa8 a and hspa8 b,hspa4a and hspa4 b were present after the divergence between teleost and Tetrapods.(5)Among various fish species,the hspa8 a gene in zebrafish has a separate sub-cluster,while hspa8 a and hspa8 b in S.marmoratus were closely related,which suggests that they duplicated after the divergence of S.marmoratus and zebrafish.In addition,analysis of the expression profile of the HSP70 family genes of S.marmoratus under temperature stress found that all 15 HSP70 family genes were expressed in the liver.Nine genes were significantly up-regulated and no significantly downregulated genes were found under high-temperature treatment,while only two genes were significantly down-regulated and no significantly up-regulated genes were found under lowtemperature treatment,indicating that the different expression patterns of HSP70 family genes may be attributed to the different influence of cold and heat on physiology.Six differentially expressed HSP70 family genes were randomly selected for quantitative realtime PCR(qRT-PCR)verification,and it was found that the expression trend of the qRTPCR results was consistent with theRNA-Seq results,indicating that the transcriptome data was highly reliable.3.Transcriptome analysis of the gills of S.marmoratus under acute salinity stressWith a salinity of 20 as the control group,the S.marmoratus was subjected to acute salinity stress at 10 and 35 salinities,and the gill tissue was taken 12 hours later for transcriptome sequencing.A total of 72.1Gb clean reads were obtained after filtering from all samples.Comparing the clean reads with the genome of S.marmoratus,the total matching degree was between 86.31% and 93.19%,indicating that the transcriptome sequencing results have high reliability.After assembly and annotation of new transcripts,2,160 of the 25,278 annotated genes were new genes.Analysis of the number of DEGs under salinity stress revealed that compared with the control group,the high-salt group and the low-salt group had 176 and 199 up-regulated genes,respectively,and 303 and 321 downregulated genes;compared with the low-salt group,there were 152 and 81 up-regulated genes and 81 down-regulated genes in the high-salt group,respectively.Among them,3genes were differentially expressed in all comparison combinations.These shared DEGs were related to cell apoptosis,allergies,and inflammatory reactions,and can play an important role in response to salinity stress.In addition,GO and KEGG enrichment analysis of DEGs found that the functions of DEGs were mainly enriched in GO entries such as metabolic processes,membranes part,protein-containing complexes,binding,ATP binding,and stress translation.Compared with the control group and the low-salt group,the differentially down-regulated genes of the high-salt group were significantly enriched in ribosome biogenesis in eukaryotes pathway,which is likely to be related to salinity stress;compared with the control group,the differentially down-regulated genes in the low-salt group were mainly enriched in the adrenergic signaling in cardiomyocytes and calcium signaling pathways.The calcium signaling pathway is believed to play an important role in ion exchange and osmotic pressure regulation;compared with the control group,the differentially up-regulated genes of the high-salt group and the low-salt group were all enriched in the metabolism of cytochrome P450 to xenobiotics and drug metabolism-other enzyme pathways,indicating that drug metabolizing enzymes play an important role in salinity stress.Six DEGs were randomly selected for qRT-PCR verification,and the results showed that the transcriptome data had high credibility.In this study,the whole transcriptome analysis under acute temperature stress and the transcriptome analysis under acute salinity stress were performed,and some important temperature and salinity regulation of key genes were found,which provided basic data for studying the molecular mechanism of temperature and salinity regulation of S.marmoratus.However,there is a lack of further expression and functional verification of the target genes of non-codingRNA under temperature stress,and the research on salinity stress has not yet reached the level of non-codingRNA.This needs to be perfected in future research in order to clarify the internal regulation mechanism of the acute temperature and salinity adaptation of S.marmoratus. |
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