MiRNA Analysis And CeRNA Regulatory Network Of Apostichopus Japonicus In Response To Vibrio Splendidus Infection And High Temperature Stress

Apostichopus japonicus,with outstanding nutritional and economic values,is one of the representative species on the fifth wave of the mariculture in China.With the breakthrough in aquatic genetic breeding technology,the sea cucumber farming has become an important support for the coastal fishery economy in China.In recent years,the development of the sea cucumber farming has been severely constrained by frequent extreme heat weather and disease disasters.Therefore,the regulatory mechanism of A.japonicus in response to infections and high temperature stress has been analyzed to lay the foundation for the disease resistance of A.japonicus,and the selection and breeding of new heat-tolerant varieties,and germplasm optimization.The research object was A.japonicus,andRNA-seq technologies were used to sequence and analyze the body wall of A.japonicus so as to obtain transcriptional expression profiles of the body wall of Acanthopanax under pathogen infestation and high temperature stress.The differentially expressed miRNA was(were)selected and verified by bioinformatics Analysis.Association analysis of differentially expressed miRNA target genes with mRNA sequencing data was carried out to screen out and validate the important negative regulatory genes and further construct miRNA-mRNA gene co-expression network;the ceRNA analysis was performed on circRNA and the circRNA-miRNA-mRNA regulatory network was constructed.This study provides basic data for the analysis of the molecular mechanism of A.japonicus in response to Vibrio splendidus infestation and high temperature stress,as well as a scientific reference for breeding new A.japonicus types are resistant to disease and high temperature.The results of the related studies are as follows:1.Correlation analysis of miRNA and mRNA in Apostichopus japonicus infected with Vibrio splendidum and construction of ceRNA regulatory networkThe body wall tissue of A.japonicus was obtained by infecting V.splendid at a concentration of1×10~6 CFU/m L,and the body wall tissue of healthy A.japonicus was used as a control for miRNA sequencing analysis.The results showed that an average of 5,902,588 and 5,053,529 valid sequences was obtained from the control group(PT10H)and the pathogen infection group(PT16S),of which70.32%of the valid sequences were between 21-24nt,and 40.32%of the sequences were 22nt in length.By database comparison,194 known miRNAs and 19 novel miRNAs were identified in the PT10H group,and 182 known miRNAs and 42 novel miRNAs were identified in the PT16S group.By differential expression analysis,2 up-regulated and 11 down-regulated significantly differentially expressed miRNAs(DEmiRNAs)were selected.The results of target gene prediction and enrichment analysis showed that DEmiRNA predicted a total of 9,680 target genes,which were annotated into8,199 GO terms,and 432 Go terms were significantly enriched,mainly related to cell signal transduction and antigen presentation.KEGG analysis showed that target genes were annotated into313 KEGG pathways,and 23 pathways were significantly enriched,mainly related to apoptosis and immune response.The 496 differentially expressed genes(DEGs)selected from the transcriptome sequencing results were correlated with DEmiRNA target genes,and a total of 40 negatively regulated DEGs were selected.The constructed miRNA-mRNA regulatory network showed that cell self-antigen isoforms(ICA69),organic cation transporter-like(ORCT)and laminin subunit beta-1(LAMB1)genes played a key role in the response of sea cucumber to V.splendidus infection.The RT-q PCR validation results showed that the expression trends of DEmiRNA and DEG were consistent with the sequencing results.In the transcriptome sequencing results,a total of 4,682 circRNAs were identified in the PT10H and PT16S groups,of which 28 circRNAs had significant differences in expression.28 differentially expressed circRNAs,13 DEmiRNAs and 40 negatively regulated DEGs were selected to construct the circRNA-miRNA-mRNA regulatory network.Enrichment analysis of genes in the ceRNA regulatory network showed that 40 genes were significantly enriched in 129 GO terms.KEGG analysis showed that 24 genes were significantly enriched in 31 pathways,and pathways related to cell activity,such as AGE-RAGE signaling pathway and m TOR signaling pathway,were significantly enriched,which may be the key pathways for A.japonicus to respond to pathogens.2.Association analysis of miRNA and mRNA in Apostichopus japonicus under high temperature stress and construction of ceRNA regulatory networkThe body wall tissue of A.japonicus was obtained at the temperature stress of 32°C,and miRNA sequencing was performed on the body wall tissue of healthy A.japonicus as the control group.The results of miRNA sequencing showed that an average of 4,847,281 valid sequences were obtained in the control group(TT10H),and a total of 263 known miRNAs and 31 novel miRNAs were identified,whereas a total of 188 known miRNAs and 17 novel miRNAs were identified in the high temperature stress group(TT30S)identified.Differential expression analysis screened 5 up-regulated and 8 down-regulated DEmiRNAs.A total of 16,563 target genes were predicted from 13 DEmiRNAs,which were annotated into 8,745 GO terms,and 334 Go terms were significantly enriched,mainly related to cell activity and cell metabolism.KEGG analysis showed that target genes were annotated into 313 KEGG pathways,and 25 pathways were significantly enriched,mainly related to cell activity and substance transport.Association analysis of DEmiRNA target genes with DEGs that obtained by mRNA sequencing screened in 101 negatively regulated DEGs.The constructed miRNA-mRNA regulatory network showed that BAG family molecular chaperone regulator 3(BAG3),E3 ubiquitin Protein ligase(TRAF7),WD repeat-containing protein 20(WDR20)and glycoprotein 3-α-L-fucosyltransferase(FUT4)genes played a key role in the response of sea cucumber in response to high temperature stress.The RT-q PCR validation results showed that the expression trends of all DEmiRNAs and DEGs were consistent with the sequencing results.A total of 6,494 circRNAs were identified in the high temperature stress group,of which 90circRNAs had significant differences in expression.Among them,75 differentially expressed circRNAs,13 DEmiRNAs and 101 negatively regulated DEGs were selected to construct a circRNA-miRNA-mRNA regulatory network.Enrichment analysis of genes in this ceRNA regulatory network showed that 101 genes were enriched into 195 GOterms.KEGG analysis showed that 58 genes were significantly enriched into 20 pathways,of which the IN17 signaling pathway and other pathways related to inflammation were significantly enriched.The results of this study showed that miRNA,cicrRNA and ceRNA played a role in the response of Apostichopus japonicus to stress.Apostichopus japonicus can regulate the expression of immune and heat stress-related genes through miRNA expression in response to Vibrio splendidus infestation and high temperature stress.The construction of the ceRNA regulatory network is indicative for the discovery and screening of key molecules in the network and the research on the regulatory network,which can lay the foundation for the subsequent functional studies of related molecules.The ubiquitin-mediated proteolysis played a key role under both stresses,but the specific regulatory mechanism further needs to be studied.