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Front Genet
2022 May 13;13:876308. doi: 10.3389/fgene.2022.876308.
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Expression Regulation Mechanisms of Sea Urchin (Strongylocentrotus intermedius) Under the High Temperature: New Evidence for the miRNA-mRNA Interaction Involvement.
Han L
,
Quan Z
,
Wu Y
,
Hao P
,
Wang W
,
Li Y
,
Zhang X
,
Liu P
,
Gao C
,
Wang H
,
Wang L
,
Zhang W
,
Yin D
,
Chang Y
,
Ding J
.
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In the context of global warming and continuous high temperatures in the northern part of China during summer, the mortality rate of our main breeding species, Strongylocentrotus intermedius, reached 80% in 2020. How sea urchins respond to high temperatures is of great concern to academia and industry. In this study, we examined the antioxidant enzyme activities of different color tube-footed sea urchins under heat stress and compared their transcriptome and microRNA (miRNA) profiles using RNA-Seq. The results showed that the antioxidant enzyme activities of sea urchins were altered by thermal stress, and the changes in peroxidase activities of red tube-footed sea urchins were particularly significant. Investigations revealed that 1,079 differentially expressed genes (DEGs), 11 DE miRNAs, and 104 "DE miRNA-DEG" pairs in total were detected in sea urchins under high temperature stress. Several mRNA and miRNAs were significantly changed (e.g. HSP70, DnaJ11, HYAL, CALR, miR-184-p5, miR-92a, miR-92c, and miR-124-p5), suggesting these genes and miRNAs exerted important functions in response to high temperature. At the transcriptional level, red tube-footed sea urchins were found to be more sensitive to high temperature and could respond to high temperature rapidly. DE miRNA-mRNA network showed that miR-92b-3p and PC-5p-7420 were the most corresponding miRNAs. Five mRNAs (DnaJ11, SAR1B, CALR, HYOU1, TUBA) may be potential markers of sea urchin response to high temperature. Possible interaction between miRNA-mRNA could be linked to protein folding in the endoplasmic reticulum, Phagosomes, and calcium transport. This study provides a theoretical basis for the molecular mechanism of sea urchin heat tolerance and information that will aid in the selection and breeding of sea urchins with high temperature tolerance.
FIGURE 1. Experimental design diagram: Molecular mechanism of high temperature tolerance of sea urchins.
FIGURE 2. Antioxidant enzyme activity determination. (A) GSH-Px activity, (B) CAT activity, (C) POD activity, (D) SOD activity. The results are expressed as mean ± SEM (n = 3). Statistical analyses of qRT-PCR data were analyzed with independent t tests. * Significant differences at p < 0.05 vs control (25 vs. 15°C), ** Highly significant differences at p < 0.01 vs control (25 vs. 15°C).
FIGURE 3. Number of DEGs in the three comparison groups.
FIGURE 4. GO enrichment of DEGs. The x-axis is the gene functional classification of GO. The y-axis is the percent of DEGs in total genes in a GO term. (A) HR vs. NR, (B) HW vs. NW, (C) HR vs. HW, (D) NR vs. NW, (E) HR vs. NW, (F) HW vs. NR.
FIGURE 5. KEGG enrichment of DEGs. The x-axis is the rich factor, which means that the proportion of DEGs in total genes in a KEGG term. The y-axis is the gene functional classification of KEGG. Various colors of plots indicate different values of −log 10 (p-value). Plot diameter represents DEG numbers in a KEGG term (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.). (A) HR vs. NR, (B) HW vs. NW, (C) HR vs. HW, (D) NR vs. NW, (E) HR vs. NW, (F) HW vs. NR.
FIGURE 6. Heat map of three groups of DE miRNA clustering analysis. The x-axis are samples and the y-axis are miRNAs. Different colors indicate different miRNA expression levels, and colors from blue via white to red indicate expression (log10 (norm value)) from low to high. Red color indicates high expression miRNA and dark blue color indicates low expression miRNA. (A) HR vs. NR, (B) HW vs. NW, (C) HR vs. HW, (D) HR vs. NW, (E) HW vs. NR.
FIGURE 7. GO annotation of DE miRNA target genes in each comparison group. The top 10 GO terms were shown in each class. (A) HR vs. NR, (B) HW vs. NW, (C) HR vs. HW, (D) NR vs. NW, (E) HR vs. NW, (F) HW vs. NR.
FIGURE 8. Enrichment analysis of KEGG pathway of DE miRNA targets in each comparison group. The top 20 enriched pathways were shown in each graph. (A) HR vs. NR, (B) HW vs. NW, (C) HR vs. HW, (D) NR vs. NW, (E) HR vs. NW, (F) HW vs. NR.
FIGURE 9. The network of DE miRNA and their target DEGs was constructed using Cytoscape 3.3.0. Yellow hexagon is miRNA, gray hexagon is target DEGs, red represents up-regulation, blue represents down-regulation.
FIGURE 10. The integrating biomolecular interaction network of target DEGs predicted to be regulated by DE miRNA in the “protein processing in endoplasmic reticulum (ko04141)” and “phagosome (ko04145)” pathways. Red diamond is KEGG pathway, yellow hexagon is miRNA, gray hexagon is target DEGs, red represents up-regulation, blue represents down-regulation.
FIGURE 11. Analysis of selected 20 differentially expressed genes (DEGs) in red and white tube-footed S. intermedius under the high temperature stress by sequencing and qRT-PCR. Each vertical bar represents the Mean ± SD (n = 3), 18s rRNA were used as a reference gene. *Significant differences at p < 0.05 vs. control (NR and NW). **Highly significant differences at p < 0.01 vs. control (NR and NW).
FIGURE 12. qRT-PCR verification of select 9 DEMs. Each vertical bar represents the Mean ± SD (n = 3), 18s rRNA were used as a reference gene. *Significant differences at p < 0.05 vs. control (NR or NW). **Highly significant differences at p < 0.01 vs. control (NR or NW).