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Identification and expression of a putative LPS-induced TNF-α factor from Asiatic hard clam Meretrix meretrix

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Abstract

LPS-induced TNF-α (LITAF) is a novel transcriptional factor that mediates the expression of inflammatory cytokines in LPS-induced processes. In the present study, the full-length cDNA encoding LITAF (designated as Mm-LITAF) was identified from Asiatic hard clam, Meretrix meretrix, by expressed sequence tag and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Mm-LITAF was 1653 bp, consisting of a 5′ untranslated region (UTR) of 91 bp, a 3′UTR of 1166 bp with one cytokine RNA instability motif (ATTTA) and one polyadenylation signal (AATAAA), and an open reading frame (ORF) of 396 bp encoding a polypeptide of 131 amino acids with a theoretical isoelectric point of 7.49, and predicted molecular weight of 14.47 kDa. The deduced amino acid of Mm-LITAF shared 29–63% similarity with the LITAFs from other species, indicating that Mm-LITAF should be a member of the LITAF family. Two highly conserved CXXC motifs forming a compact Zn2+-binding structure were also identified in Mm-LITAF. A quantitative reverse transcriptase real-time PCR (qRT-PCR) assay was developed to assess the expression of Mm-LITAF mRNA in different tissues, and the temporal expression of Mm-LITAF in clams challenged with Vibrio anguillarum. The mRNA transcript of Mm-LITAF could be detected in all the examined tissues with the highest expression level in the gill. Mm-LITAF expression was up-regulated significantly at 16 h in the gill and at 8 h in haemocytes after bacterial challenge, respectively. These results suggest that the Mm-LITAF is a constitutive and inducible acute-phase protein that perhaps involved in the innate immune response of hard clam.

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Acknowledgments

This work was supported by Liaoning Scientific Research Program of Ocean and Fisheries department (200801) and Dalian Science & Technology Foundation (2010J21DW033).

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Authors and Affiliations

  1. Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fishery Science Institute, Dalian, 116023, China

    Hong-jun Li

  2. National Marine Environmental Monitoring Center, Dalian, 116023, China

    Qing Yang

  3. Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fishery Science Institute, Dalian, 116023, China

    Xiang-gang Gao, Hao Su, Juan Wang & Chong-bo He

Authors
  1. Hong-jun Li
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  2. Qing Yang
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  3. Xiang-gang Gao
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  4. Hao Su
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  5. Juan Wang
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  6. Chong-bo He
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Correspondence to Chong-bo He.

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11033_2011_810_MOESM1_ESM.doc

Fig. 1 Amino acid alignment of Mm-LITAF with other known LITAF sequences. Amino acid residues that are conserved in at least 50% sequences are shaded in dark and similar amino acids are shaded in grey. Eight conserved cysteines are labeled by inverted filled traingle; two CXXCXXC motifs are indicated at the bottom. The origin and accession number for each of the retrieved LITAF sequences in this study were showed in Table 2 (Supplement). (DOC 33 kb)

Table 1 Primers used in this study (DOC 485 kb)

Table 2 LITAF amino acid identity determined by the ClustalW (DOC 30 kb)

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Li, Hj., Yang, Q., Gao, Xg. et al. Identification and expression of a putative LPS-induced TNF-α factor from Asiatic hard clam Meretrix meretrix . Mol Biol Rep 39, 865–871 (2012). https://doi.org/10.1007/s11033-011-0810-6

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