MOLECULAR CLONING, CHARACTERIZATION, AND EXPRESSION ANALYSIS OF LIPOPOLYSACCHARIDE-INDUCED TNF-alpha FACTOR (LITAF) IN THE MUD CRAB, SCYLLA PARAMAMOSAIN

摘要

Lipopolysaccharide-induced TNF-alpha factor (LITAF) is an important transcription factor mediating the expression of inflammatory cytokines, including TNF-alpha, which is involved in the innate immune defence against infectious agents. In this study, the Scylla paramamosain LITAF (SpLITAF) gene was cloned and identified from S. paramamosain using RT-PCR and RACE. The full length cE)NA of SpLITAF was 1116 bp, contained an ORF of 495 bp encoding a polypeptide of 164 amino acids with predicted molecular mass of 17.86 kDa and a theoretical isoelectric point of 8.51. Sequence alignment showed that SpLITAF possessed a typical LITAF domain with two CXXC motifs and eight conserved cysteine residues. Phylogenetic analysis indicated that SpLITAF clustered closely with known LITAFs from Eriocheir sinensis and Litopenaeus vannamei. qRT-PCR analysis showed SpLITAF mRNA transcript expressed ubiquitously in all examined tissues with the highest expression in haemocytes, while lower expression in hepatopancreas. Its expression levels displayed a trend of up-regulation and then returned to its initial value in haemocytes and gill, post challenge with LPS, Poly (I : C), and Vibrio alginolyticus, respectively. Furthermore, the SpLITAF protein was verified to be located in hyalinocytes, semi-granulocytes and granulocytes, and was distributed throughout the cytoplasm and nucleus, especially in cytoplasm. These results provide a basis for further investigating the roles of SpLITAF in the immune signaling pathways as a transcription factor in S. paramamosain.