RESEARCH ARTICLE The Effect of NF-κB Signalling Pathway on Expression and Regulation of Nacrein in Pearl Oyster, Pinctada fucata

Nacrein is the first identified and widely investigated molluscan matrix protein and is consid-ered to play an important role in the shell formation of the pearl oyster, Pinctada fucata. Here, we investigate the effect of the NF-κB signalling pathway on Nacrein gene expression in P. fucata to elucidate the mechanisms involved in shell formation. Inhibition of NF-κB signalling decreased Nacrein promoter-dependent luciferase activity. However, co-trans-fection of the Nacrein promoter vector with Pf-IKK or Pf-Rel expression plasmids could enhance luciferase activity, thus proving NF-κB signalling could regulate the transcriptional activity of the Nacrein promoter. Gene silencing by RNA interference and subsequent observation of the inner surface of the nacreous layer of oyster shells by SEM, showed that suppression of the gene Pf-Rel lead to a partial inhibition of Nacrein expression, not only at the mRNA level but also at the protein level. The inner surface of the shells became abnor-mal. Electrophoretic mobility shift assays (EMSAs) revealed that Pf-Rel could directly bind to the relative sites of the Nacrein promoter. These results confirm that an important compo-nent of the NF-κB signalling pathway, Pf-Rel, can directly bind the Nacrein promoter in P. fucata, and regulate its transcription and shell formation