Members of the TRAF gene family in Octopus sinensis and their response to PGN, poly I:C, and Vibrio parahaemolyticus.

Octopus sinensis, the species of Cephalopoda, is known as the highest Mollusca and is an economic and new aquaculture species in the coastal waters of southern China. The immune system has been well documented to have a function of resisting the invasion of pathogens in the external environment among mollusca species. As a kind of signaling molecule in the innate immune system, tumor necrosis factor (TNF) receptor-associated factor (TRAF) plays significant roles in TNF receptor (TNFR)/interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) signaling pathways. Until now, seven TRAF members (TRAF1-7) have been discovered, and they have been reported to participate in regulating signal pathways mediated by pattern recognition receptors and play important roles in the innate immune response of the hosts. In this study, five TRAF genes of O. sinensis (OsTRAF2, OsTRAF3, OsTRAF4, OsTRAF6, and OsTRAF7) were identified, whose full length of the open reading frame is 1473 bp, 1629 bp, 1431 bp, 1353 bp and 2121 bp respectively, encoding 490, 542, 476, 450 and 706 amino acids, respectively. Bioinformatics analysis showed that each OsTRAF has different chromosome locations. In addition to seven consecutive WD40 domains on the C-terminal of OsTRAF7 protein, the C-terminal of OsTRAF proteins all contain a conserved TRAF domain, namely the MATH domain. Phylogenetic analysis showed that OsTRAF proteins were clustered together with TRAF proteins of bivalves. Moreover, TRAF1 and TRAF2, TRAF3 and TRAF5 were clustered together in a large clade, respectively, revealing they have a close genetic relationship. The results of quantitative Real-time PCR showed that OsTRAF genes were highly expressed in the gill, hepatopancreas and white body. After stimulation with PGN, poly I:C and V. parahaemolyticus, the expression levels of OsTRAF genes were up-regulated in the gill, hepatopancreas and white body at different time points. These results indicated that OsTRAF genes play an important role in the antibacterial and antiviral immune response of O. sinensis.

Genome-wide identification of toll-like receptors in Octopus sinensis and expression analysis in response to different PAMPs stimulation.

Toll-like receptors (TLRs) are one of the extensively studied pattern recognition receptors (PRRs) and play crucial roles in the immune responses of vertebrates and invertebrates. In this study, 14 TLR genes were identified from the genome-wide data of Octopus sinensis. Protein structural domain analysis showed that most TLR proteins had three main structural domains: extracellular leucine-rich repeats (LRR), transmembrane structural domains, and intracellular Toll/IL-1 receptor domain (TIR). The results of subcellular localization prediction showed that the TLRs of O. sinensis were mainly located on the plasma membrane. The results of quantitative real-time PCR (qPCR) showed that the detected TLR genes were differentially expressed in the hemolymph, white bodies, hepatopancreas, gills, gill heart, intestine, kidney, and salivary gland of O. sinensis. Furthermore, the present study investigated the expression changes of O. sinensis TLR genes in hemolymph, white bodies, gills, and hepatopancreas in different phases (6 h, 12 h, 24 h, 48 h) after stimulation with PGN, poly(I: C) and Vibrio parahaemolyticus. The expression of most of the TLR genes was upregulated at different time points after infection with pathogens or stimulation with PAMPs, a few genes were unchanged or even down-regulated, and many of the TLR genes were much higher after V. parahaemolyticus infection than after PGN and poly(I:C) stimulation. The results of this study contribute to a better understanding of the molecular immune mechanisms of O. sinensis TLRs genes in resistance to pathogen stimulation.

The immune function of TLR4-1 gene in Octopus sinensis revealed by RNAi and RNA-seq.

Toll-like receptors (TLRs) are a class of conserved pattern recognition receptors (PRRs) that are crucial for initiating the innate immune response and aiding in the clearance of pathogenic organisms. Many studies have identified TLR4 as a distinctive member of the TLR family, capable of activating both the Myeloid differentiation factor 88-dependent signaling pathway (MyD88-dependent) and the TIR-domain-containing adaptor inducing IFN-ß dependent signaling pathway (TRIF-dependent). Nevertheless, the role of TLR4 in Cephalopoda is still largely unexplored. To elucidate the immune function of the OsTLR4-1 gene in Octopus sinensis, the OsTLR4-1 gene was first validated and analyzed in this study. The cDNA comprises a 2475 bp ORF region, encoding 824 amino acids. Evolutionary tree analysis indicated a high homology and a close phylogenetic relationship between the Octopus sinensis and other mollusks. RNA interference (RNAi) experiments demonstrated that the expression level of OsTLR4-1 gene and its protein in the lymphocytes of the RNAi group treated with OsTLR4-1 dsRNA was extremely significantly lower than that of the blank control group and negative control group (P < 0.01), and the expression of downstream genes of OsTLR4-1, including ligand MyD88, IRAK4, TRAF6, MKK6, Hsp90, COX2, TRAF3, and RIP1, were significantly down-regulated compared to the blank and negative control group (P < 0.01). Additionally, OsTLR4-1 expression in lymphocytes was highly significantly up-regulated in the LPS-treated group compared to the blank control group (P < 0.01), while its expression was extremely significantly lower in the LPS-treated group after OsTLR4-1 interference than in the blank control group (P < 0.01). The expression of its downstream effector genes Big Defensin (Big-Def) and histone H2A.V (H2A.V) was highly significantly up-regulated in lymphocytes in the LPS-treated group compared to the blank control group (P < 0.01), while their expression in the LPS-treated group after OsTLR4-1 interference was extremely significantly lower than that in the blank control group (P < 0.01). Through comparative transcriptome analysis of the RNAi group and the blank control group, it was found that differentially expressed genes were enriched in the Toll-like receptor signaling pathway, PI3K-AKT signaling pathway, P53 signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. qRT-PCR results of key genes in these pathways revealed a decrease in all genes except IκB and Jun2 genes. This study enhances our understanding of the immune function of the TLR gene family in O. sinensis and provides a foundation for further research into innate immune signaling pathways in cephalopods.

Transcriptome analysis provides insights into differentially expressed genes and long noncoding RNAs involved in sex-related differences in Amur sturgeon (Acipenser schrenckii).

In the present study, the next-generation sequencing technology was used to develop a transcriptome database of gonad and liver from 3-year-old male and female Amur sturgeons (Acipenser schrenckii). A total of 139,406 unigenes were generated after the Illumina Hiseq. 2500 sequence and assembled by Trinity. The differential expression analysis between male and female obtained 5,199 differentially expressed genes (DEGs) in gonad and 457 DEGs in liver. Gene Ontology enrich analysis showed that the specific DEGs of gonad play a dominant role in reproductive processes. Although the specific DEGs of liver indicated their primary responsibility for energy metabolism, the DEGs of liver and gonad co-own enriched in terms associated with reproduction suggested that liver also plays a role in sex-related differences in Amur sturgeon. Furthermore, genes related to sex-related differences were selected to validate among the four different tissues by real-time quantitative polymerase chain reaction (qRT-PCR). In addition, by trans-acting analysis, a total of 5,206 putative long noncoding RNAs (lncRNAs) and 3,490 target genes of lncRNAs were predicted from gonad and liver. Moreover, several lncRNAs targeting Mea1, Piwil1, Tdrd1, Nanos2, Ankrd49, and ZP3 may have potential regulatory effect related to gametogenesis and gonadal differentiation were identified and validated by qRT-PCR. These results suggested for the first time that lncRNAs might be one of the effect factors in regulating the differential expression of messenger RNAs associated with sex-related differences in Amur sturgeon.