A putative endocrine factor SIBD (single insulin binding domain protein) involved in immune response of Chinese mitten crab Eriocheir sinensis.

Insulin growth factor binding proteins (IGFBPs), characterized by the conserved insulin binding (IB) domains, are important components of endocrine system and play key roles in metabolism and growth. In the present study, the full-length cDNA of a single IB domain protein (designated EsSIBD) was identified from Chinese mitten crab Eriocheir sinensis based on expressed sequence tag (EST) sequence. The 1187 bp EsSIBD cDNA contained a 321 bp open reading frame (ORF) encoding a putative protein of 106 amino acids, a 5'-untranslated region (UTR) of 189 bp, and a 3'-UTR of 677 bp. Multiple sequence alignment presented ten conserved cysteine residues critical for the fundamental structure and function of IB domain. BLAST analysis revealed that EsSIBD shared high similarity with previously known IB domains of IGFBPs with the identities ranging from 40% to 46%. The sequence similarity and domain conservation indicated that EsSIBD was a potential member of the IGFBP family. Phylogenic analysis presented that EsSIBD was closer to IGFBP7 than to the other IB domain containing proteins, suggesting its functional similarity with the endocrine factor IFGBP7. The mRNA expression of EsSIBD in different tissues including hepatopancreas, gill, gonad, muscle, heart and haemocytes, and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EsSIBD mRNA transcripts could be detected in all examined tissues with the highest expression level in gill. The EsSIBD mRNA expression in haemocytes was sensitive to L. anguillarum stimulation and it was up-regulated from 3 to 24 h after challenge. The structure conservation and functional similarity to IFGBPs, and its sensitivity to L. anguillarum stimulation collectively implied that EsSIBD was probably involved in endocrine and immune systems of Chinese mitten crab, and provided insight into the cross-talk between the invertebrate endocrine and immune system.

Gai Y ，Wang L ，Song L ，Zhao J ，Qiu L ，Xing K ... -  《-》

A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression.

Gai Y ，Qiu L ，Wang L ，Song L ，Mu C ，Zhao J ，Zhang Y ，Li L ... -  《-》

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Gai Y ，Zhao J ，Song L ，Li C ，Zheng P ，Qiu L ，Ni D ... -  《FISH & SHELLFISH IMMUNOLOGY》

Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis.

NF-κB is a B-cell specific transcription factor that plays crucial roles in inflammation, immunity, apoptosis, development and differentiation. In the present study, a novel NF-κB-like transcription factor Relish was cloned from Chinese mitten crab Eriocheir sinensis (designated as EsRelish) by rapid amplification of cDNA ends (RACE) technique based on expressed sequence tag (EST). The full-length cDNA of EsRelish was of 5034 bp, consisting of a 5' untranslated region (UTR) of 57 bp, a 3' UTR of 1335 bp with two mRNA instability motifs (ATTTA), a polyadenylation signal sequence (AATAAA) and a poly (A) tail, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids with a calculated molecular mass of 134.8 kDa and a theoretical isoelectric point of 5.26. There were a typical Rel homology domain (RHD), two nuclear localization signal (NLS) sequences (KR), an inhibitor κB (IκB)-like domain with six ankyrin repeats, a PEST region and a death domain in the deduced amino acid sequence of EsRelish. Conserved domain, higher similarity with other Rel/NF-κBs and phylogenetic analysis suggested that EsRelish was a member of the NF-κB family. Quantitative real-time RT-PCR was employed to detect the mRNA transcripts of EsRelish in different tissues and its temporal expression in hemocytes of E. sinensis challenged with Pichia methanolica and Listonella anguillarum. The EsRelish mRNA was found to be constitutively expressed in a wide range of tissues. It could be mainly detected in the hemocytes, gonad and hepatopancreas, and less degree in the gill, muscle and heart. The expression level of EsRelish mRNA in hemocytes was up-regulated from at 3, 6, 9 and 12 h after P. methanolica challenge. In L. anguillarum challenge, it was up-regulated at 9, 12 and 24 h. The results collectively indicated that EsRelish was potentially involved in the immune response against fungus and bacteria.

Li F ，Wang L ，Zhang H ，Zheng P ，Zhao J ，Qiu L ，Zhang Y ，Song L ... -  《-》

A γ-aminobutyrate type A receptor-associated protein involved in the immune response of Eriocheir sinensis.

The γ-aminobutyrate type A receptor-associated protein (GABARAP) is a ubiquitin-like modifier, which is implicated in membrane trafficking and fusion events of γ-aminobutyrate type A receptor, autophagy and apoptosis. In the present study, the gene encoding GABARAP (designated EsGABARAP) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends approach and expression sequence tag (EST) analysis. The full-length cDNA of EsGABARAP was of 457 bp, containing a 5' untranslated region (UTR) of 77 bp, a 3' UTR of 32 bp with a poly(A) tail and an open reading frame (ORF) of 348 bp encoding a polypeptide of 116 amino acids with the predicted molecular weight of 13.81 kDa and theoretical isoelectric point of 8.73. The deduced amino acid sequence of EsGABARAP shared higher similarity (91.8-97.4%) with those of other GABARAPs, and it contained a conserved MAP1_LC3 domain. In the phylogenetic tree, EsGABARAP was firstly clustered with GABARAPs from other animals and then gathered together with the same family proteins of GABARAP. The mRNA expression level of EsGABARAP in six tissues and its temporal expression level in haemocytes of crabs challenged with Listonella anguillarum were determined by quantitative real-time RT-PCR. The mRNA transcripts of EsGABARAP could be detected ubiquitously in the examined tissues, including haemocytes, hepatopancreas, muscle, gill, heart and gonad, with the highest expression level in hepatopancreas. The expression level of EsGABARAP mRNA in haemocytes was up-regulated after L. anguillarum challenge and reached 6.58-fold of that in blank group at 24 h (P < 0.05) and 7.52-fold at 48 h (P < 0.05). The increasing transcripts in haemocytes after L. anguillarum challenge gave the preliminary evidence for the involvement of EsGABARAP as a part of immune response against bacteria challenge in crabs.

Zhou Z ，Wang L ，Kong P ，Qiu L ，Zhang H ，Gao Y ，Yang J ，Song L ... -  《-》