Identification and Characterization of MicroRNAs from Longitudinal Muscle and Respiratory Tree in Sea Cucumber (Apostichopus japonicus) Using High-Throughput Sequencing

Abstract

MicroRNAs (miRNAs), as a family of non-coding small RNAs, play important roles in the post-transcriptional regulation of gene expression. Sea cucumber (Apostichopus japonicus) is an important economic species which is widely cultured in East Asia. The longitudinal muscle (LTM) and respiratory tree (RPT) are two important tissues in sea cucumber, playing important roles such as respiration and movement. In this study, we identified and characterized miRNAs in the LTM and RPT of sea cucumber (Apostichopus japonicus) using Illumina HiSeq 2000 platform. A total of 314 and 221 conserved miRNAs were identified in LTM and RPT, respectively. In addition, 27 and 34 novel miRNAs were identified in the LTM and RPT, respectively. A set of 58 miRNAs were identified to be differentially expressed between LTM and RPT. Among them, 9 miRNAs (miR-31a-3p, miR-738, miR-1692, let-7a, miR-72a, miR-100b-5p, miR-31b-5p, miR-429-3p, and miR-2008) in RPT and 7 miRNAs (miR-127, miR-340, miR-381, miR-3543, miR-434-5p, miR-136-3p, and miR-300-3p) in LTM were differentially expressed with foldchange value being greater than 10. A total of 14,207 and 12,174 target genes of these miRNAs were predicted, respectively. Functional analysis of these target genes of miRNAs were performed by GO analysis and pathway analysis. This result provided in this work will be useful for understanding biological characteristics of the LTM and RPT of sea cucumber and assisting molecular breeding of sea cucumber for aquaculture.

Fig 1. Common and specific unique reads obtained by deep sequencing of small RNA transcriptome in the longitudinal muscle (LTM) and respiratory tree (RPT).

The pink denotes the specific unique reads from RPT, the green denotes the specific unique reads from LTM, and the purple denotes the common unique reads obtained from both LTM and RPT.

Fig 2. Length distribution of small RNAs identified from the LTM (A) and RPT (B) of sea cucumber (A. japonicus).

Fig 3. Common and specific conserved miRNAs in the longitudinal muscle (LTM) and respiratory tree (RPT).

The blue denotes the specific conserved miRNAs identified from LTM. The pink denotes specific conserved miRNAs identified in RPT, and the purple denotes the common conserved miRNAs in both LTM and RPT.

Fig 4. Differential expression of miRNAs between LTM and RPT.

miRNAs with foldchanges between -10 and 10 were highlighted in red; foldchanges between -2 and -10 or between 2 and 10 were highlighted in green; and foldchanges between -2 and 2 were highlighted in purple.

Fig 5. Validation of expression analysis of miRNAs using qRT-PCR.

Purple bars indicate the expression in the LTM tissue, while dark red bars indicate the expression in the tissue of RPT.

Fig 6. GO analysis for predicted target genes of differentially expressed miRNAs.

The dark red bars indicate the gene numbers/percentages within each GO term at level 2 in the LTM, and the grey bars indicated the gene numbers/percentages within each GO term at level 2 in the RPT.

Fig 7. Common and shared target gene KEGG pathways between LTM and RPT.

The blue indicates the specific pathways in the LTM, the pink indicates the specific pathways in the RPT, and the dark red indicates the common pathways shared by LTM and RPT.