Abstract
Skin ulceration syndrome (SUS) is considered to be a major constraint for the stable development of Apostichopus japonicus culture industries. In this study, we investigated protein changes in the coelomocytes of A. japonicus challenged by Vibrio splendidus using isobaric tags for relative and absolute quantification (iTRAQ) over a 96 h time course. Consequently, 228 differentially expressed proteins were identified in two iTRAQs. A comparison of the protein expression profiles among different time points detected 125 proteins primarily involved in response to endogenous stimuli at 24 h. At 48 h, the number of differentially expressed proteins decreased to 67, with their primary function being oxidation reduction. At the end of pathogen infection, proteins responsive to amino acid stimuli and some metabolic processes were classified as the predominant group. Fifteen proteins were differentially expressed at all time points, among which eight proteins related to pathologies in higher animals were shown to be down-regulated after V. splendidus infection: paxillin, fascin-2, aggrecan, ololfactomedin-1, nesprin-3, a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (Adamts7), C-type lectin domain family 4 (Clec4g) and n-myc downstream regulated gene 1 (Ndrg1). To gain more insight into two SUS-related miRNA (miR-31 and miR-2008) targets at the protein level, all 129 down-regulated proteins were further analyzed in combination with RNA-seq. Twelve and eight proteins were identified as putative targets for miR-31 and miR-2008, respectively, in which six proteins (5 for miR-31 and 1 for miR-2008) displayed higher possibilities to be regulated at the level of translation. Overall, the present work enhances our understanding of the process of V. splendidus-challenged sea cucumber and provides a new method for screening miRNAs targets at the translation level.
Highlights
Sea cucumber Apostichopus japonicus, famous for its superior nutritive value and supposed medicinal properties, has become one of the most important aquaculture species in China, and its aquaculture has grown rapidly since the 1980s
Since miRNAs can function through partial or full complementary base pairing to their target messenger RNAs, resulting in PLOS ONE | www.plosone.org isobaric tags for relative and absolute quantification (iTRAQ)-Based Proteomics in Apostichopus japonicus translational inhibition or mRNA degradation [12], it seems to be feasible to identify the putative targets of miRNAs by detecting the cellular level of mRNAs or proteins from the same biological sample
Negative expression correlations were not detected by qRT-PCR for most of their putative targets at the mRNA level in bacteria challenged samples, indicating that the targets might be regulated at the protein level without affecting mRNA abundance
Summary
Sea cucumber Apostichopus japonicus, famous for its superior nutritive value and supposed medicinal properties, has become one of the most important aquaculture species in China, and its aquaculture has grown rapidly since the 1980s. Since miRNAs can function through partial or full complementary base pairing to their target messenger RNAs (mRNAs), resulting in PLOS ONE | www.plosone.org iTRAQ-Based Proteomics in Apostichopus japonicus translational inhibition or mRNA degradation [12], it seems to be feasible to identify the putative targets of miRNAs by detecting the cellular level of mRNAs or proteins from the same biological sample. To understand the intrinsic pathogenic mechanism, we analyzed protein expression patterns over a 96 h time course pathogen infection to identify proteins and peptides changes in response to V. splendidus challenge and reveal miRNA targets at the translational level, increasing our knowledge of cellular pathways important for infection and pathogenesis
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