Pengzhi Qi

Identification and comparative analysis of the Pseudosciaena crocea microRNA transcriptome response to poly(I:C) infection using a deep sequencing approach.

Two sRNA libraries with or without poly(I:C) infection of large yellow croaker Pseudosciaena crocea were constructed and sequenced using the high-throughput Illumina/Solexa deep sequencing technology. The high-throughput sequencing pipeline yielded 163,79,272 and 217,07,070 raw reads corresponding to 132,27,594 and 206,86,409 clean reads for the normal and infected libraries, respectively. Bioinfromatic analysis identified 534 miRNAs, of which, 158 miRNAs were known in miRBase 20.0 and the remaining 376 were not found homology to any known metazoan miRNAs, suggesting a possible species-specificity. We analyzed the significance of differently expressed miRNAs between two libraries using pairwise comparison. There was significant differential expression of 112 miRNAs (p < 0.001) between two libraries. Thereinto, a number of known miRNAs were identified immune-related. Real-time quantitative PCR experiments (RT-qPCR) were preformed for 6 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first comprehensive study of miRNAs in P. crocea and of expression analysis of P. crocea miRNAs in response to poly(I:C) infection, and many miRNAs were differentially regulated under normal and infection conditions. These findings deepened our understanding of the role of miRNAs in the intricate hosts immune system, and should be useful to develop new control strategies for host immune defense against various foreign infection in P. crocea.

Molecular characterization and expression analysis of a complement component C3 in large yellow croaker (Larimichthys crocea)

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. Complement component 3 is a key molecule in the complement system, whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the large yellow croaker (Larimichthys crocea), which was high similarity to other complement C3. We reported the primary sequence, tissue expression profile, polypeptide domain architecture and phylogenetic analysis of L. crocea complement component C3 (L.c-C3) gene. Its open reading frame (ORF) is 4962 bp and encodes for 1653 amino acids with a putative signal peptide of 23 amino acid residues. The deduced amino acid sequence showed that L.c-C3 has conserved residues and domains known to be crucial for C3 function. Phylogenetic analysis showed that L. crocea was closely related to Miichthys miiuy. The mRNA expressions of L.c-C3 was detectable at different tissues. L.c-C3 was expressed in a wide range of adult tissues, it showed highest expression in the liver. But the different developmental stages from fertilized egg to newborn larvae of the large yellow croaker the highest expression levels of L.c-C3 gene were not found. Bacterial challenge experiments showed that the levels of L.c-C3 mRNA expression were up-regulated in the liver, spleen and brain of adult large yellow croaker respectively. The results showed that L.c-C3 mRNA expression in the large yellow croaker is influenced by bacterial stress and L.c-C3 might play an important role in immunity mechanisms. This study will further increase our understanding of the function of L.c-C3 and molecular mechanism of innate immunity in teleosts.

Transcriptome analysis of Mytilus coruscus hemocytes in response to Vibrio alginnolyficus infection

ABSTRACT As an economically important bivalve, the Mytilus coruscus is cultured widely in the eastern coast of China. In recent years, this bivalve has been seriously affected by the pathogenic infections. To elucidate the host defense mechanisms of M. coruscus against pathogenic challenge, the hemocyte transcriptomes of M. coruscus before and after Vibrio alginnolyficus infection were analyzed using the deep‐sequencing platform Illumina/HiSeq‐2500, meanwhile the differentially expressed genes (DEGs) were investigated. In total, 130,031,083 clean reads were obtained and then assembled into 63,942 unigenes with an average length of 810 bp and an N50 of 1056 bp. Unigenes were annotated by comparing against nr, Swiss‐Prot, KEGG, COG, KOG, GO, and Pfam databases, and 27,345 unigenes (42.77%) were annotated in at least one database. After bacterial challenge, 1270 and 265 genes were identified as remarkably up‐ or down‐regulated, respectively, amongst 1154 were associated with 122 pathways, including classical immune‐related pathways, such as ‘Toll‐like receptor signaling’, ‘the complement cascades’, ‘MAPK signaling pathway’, ‘Apoptosis’ and ‘Wnt signaling pathway’. Besides, nine genes which were differently‐expressed immuno‐related were confirmed by using quantitative real‐time PCR. These findings would provide new insights on the M. coruscus innate immunity, based on which, some novel strategies for management of diseases and long‐term sustainability of M. coruscus culture could be developed. HighlightsThe sequencing libraries of M. coruscus hemocytes before and after Vibrio alginnolyficus infection were constructed.This two libraries were sequenced using the high throughput sequencing technology.The expression profiles for identified unigenes were analyzed.The selected DEGs were identified using the RT‐qPCR technology.

Molecular characterization of complement component 3 (C3) in Mytilus coruscus improves our understanding of bivalve complement system

&NA; Complement component 3 (C3) plays a central role in the complement system whose activation is essential for all the important functions performed by this system. Here, a novel C3 gene, termed Mc‐C3, was identified from thick shell mussel (Mytilus coruscus). The deduced Mc‐C3 protein possessed the characteristic structure features present in its homologs and contained the A2M_N_2, ANATO, A2M, A2M_comp, A2M_recep, and C345C domains, as well as the C3 convertase cleavage site, thioester motif, and conserved Cys, His, and Glu residues. Mc‐C3 gene constitutively expressed in all examined tissues and predominantly expressed in immune‐related tissues such as gills, hemocytes and hepatopancreas. After stimulation with lipopolysaccharide or Cu2+, the expression of Mc‐C3 was significantly induced in gills. Further luciferase reporter assays showed the ability for activation of NF‐&kgr;B signaling transduction of Mc‐C3a. Taken together, these results show that C3 may play an essential role in the immune defense of M. coruscus. The present data therefore provide a more detailed insight into the functional activities of the bivalve complement system. HighlightsThe novel complement component 3 gene was identified from M. coruscus.Mc‐C3 gene constitutively expressed in all examined tissues.Mc‐C3 was significantly induced in gills with LPS and Cu2+ stimulations.Mc‐C3a could activate the NF‐&kgr;B signaling transduction.

Complete mitochondrial genome of the spineless cuttlefish Sepiella inermis (Sepioidea, Sepiidae)

Abstract In this study, we determined the complete mitochondrial genome of the spineless cuttlefish Sepiella inermis. The genome was 16,191u2009bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 2 main non-coding regions [both are control regions (CR)]. The composition and order of genes, for the mitogenome found in S. inermis were similar to most other invertebrates. The overall base composition of S. inermis is T 35.6%, C 16.4%, A 40.0% and G 8.0%, with a highly Au2009+u2009T bias of 75.6%. Two control regions contain both termination-associated sequences and conserved sequence blocks. Thus, mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Sepiidae.

Complete mitochondrial genome of the common cuttlefish Sepia pharaonis (Sepioidea, Sepiidae)

Abstract In this study, the complete mitochondrial genome of the common cuttlefish Sepia pharaonis was determined first. The genome was 16,208u2009bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions [both are control regions (CR)], the gene composition and order of which were similar to most other invertebrates. The overall base composition of S. pharaonis is T: 36.3%, C: 14.7%, A: 40.9% and G: 8.1%, with a hightly Au2009+u2009T bias of 77.2%. Two control regions all contain termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the investigations of the phylogenetic relationships, taxonomic resolution and phylogeography of the Sepiidae.

Complete mitochondrial genome of the needle cuttlefish Sepia aculeata (Sepioidea, Sepiidae)

Abstract In this study, we determined the complete mitochondrial genome of the needle cuttlefish Sepia aculeata. The genome was 16,219u2009bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions [both are control regions (CR)]. The composition and order of genes, for the mitogenome found in S. aculeate, were similar to most other invertebrates. The overall base composition of S. aculeata is T 34.0%, C 17.0%, A 40.5% and G 8.5%, with a highly Au2009+u2009T bias of 74.5%. Two control regions (CR) both contain termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Sepiidae.

The receptor for activated C kinase 1 (RACK1) mediating immune response in thick shell mussel Mytilus coruscus

ABSTRACT The receptor for activated C kinase 1 (RACK1) is a intracellular receptor for the protein kinase C family which mediates various biological processes. Here, a novel RACK1 gene termed Mc‐RACK1 was identified from thick shell mussel, Mytilus coruscus. Mc‐RACK1 shared typical RACK1 domains containing WD repeats, PKC phosphorylation sites, N‐myristoylation sites, PKC activation sites, TK phosphorylation site and WD motifs. Mc‐RACK1 was constitutively expressed in all examined tissues, and its expression in gills, haemocytes and digestive glands were significantly up‐regulated upon LPS challenge. Mc‐RACK1 showed a significantly down‐regulated expression in gills and haemocytes at the early phase upon copper exposure. Mc‐RACK1 in haemocytes was silenced after receiving its dsRNA, meanwhile, the increases of SOD and CAT activity were investigated. Further, Mc‐RACK1 could activate the NF‐&kgr;B and ISRE reporter in HEK‐293T cells. These suggested that Mc‐RACK1 had a deeper involvement in mollusc immunity, and played an important role in antioxidant system. HighlightsA novel RACK1 gene was firstly identified from Mytilus coruscus.Mc‐RACK1 expression were significantly up‐regulated upon LPS challenge.Mc‐RACK1 expression were down‐regulated at the early phase upon copper exposure.The enhanced activities of SOD and CAT in haemocytes received Mc‐RACK1 dsRNA.Mc‐RACK1 could activate the NF‐&kgr;B and ISRE reporter in HEK‐293T cells.

A novel interleukin-1 receptor-associated kinase-4 from thick shell mussel Mytilus coruscus is involved in inflammatory response

Interleukin-1 receptor-associated kinase-4 (IRAK4) is considered as the most upstream kinase of IRAKs and plays a vital role in Toll-like receptor/Interleukin-1 receptor (TLR/IL-1R) signal transduction. In the present study, IRAK4 from thick shell mussel Mytilus coruscus (McIRAK4) was identified and characterized. McIRAK4 showed the most similarity to its counterparts in bivalves. The conserved death domain (DD) and catalytic domain of serine/threonine kinases (STKc) were predicted in all examined IRAK4s. McIRAK4 transcripts were constitutively expressed in all examined tissues with the higher expression level in immune related tissues, and were significantly induced in haemocytes upon lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly I:C) challenge. Further, the expression of McIRAK4 was obviously repressed by dsRNA mediated RNA interference (RNAi), meanwhile the proinflammatory cytokines TNF-alpha and IL17 were down-regulated while the antiinflammatory cytokine TGF-β was up-regulated. Additionally, McIRAK4 showed a global cytoplasmic localization in HEK293T cells through fluorescence microscopy. These results collectively indicated that McIRAK4 is one member of IRAK4 subfamily and might play the potential signal transducer role in inflammatory response. The present study provides supplement for TLR-mediated signaling pathway triggered by pathogenic invasions in thick shell mussel, and contributes to the clarification of the innate immune response in molluscs.

Preliminary Study of the Potential Mechanism of CTSD in the Aging Process of Sepiella japonica: Fundamental Function Analysis

Cathepsin D, a kind of endopeptidase, can degrade peptides and proteins in lysosomes, which are involved in cell apoptosis. Previous transcriptome analysis of optic glands of Sepiella japonica across four growth stages, expression of a cathepsin D-like segment was found to be significantly different. Based on the complete cDNA sequence of S. japonica, the CTSD gene (also called sjCTSD, GenBank accession no. KY745896.1) was cloned using RACE amplification; this gene is 1389 bp in length and encodes proteins composed of 393 amino acids. Spatio-temporal expression profiles of the sjCTSD gene were determined using qPCR assays, which showed that the expression levels of sjCTSD constantly increased across four growth stages in 9 of 11 tissues that were investigated. In the optic glands, as well as pancreas and liver cells, sjCTSD expression levels sharply increased during the post-spawning phase. To investigate the potential role of the sjCTSD gene in aging progress, we constructed the prokaryotic expression vector of pET28a/sjCTSD. After induced by IPTG, the recombinant protein sjCTSD was obtained in the form of inclusion bodies, with a molecular size of approximately 40.3 kDa, the inclusion body of sjCTSD can be converted to soluble protein through the denaturation and renaturation. The results of functional experiments showed that sjCTSD could degrade bovine hemoglobin under acidic conditions, and inhibit the growth of Escherichia coli and Vibrio alginolyticus, which was speculated that the increased expression of sjCTSD may help inhibit the invasion of pathogenic bacteria with the immune function of cuttlefish declines during the aging process. To a certain extent, these results indicated the potential functional role of the sjCTSD gene in the aging process of S. japonica. This study provides insights to further understand the roles of lysosomal proteins on anti-aging effects in S. japonica and other cephalopoda species.