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Nacrein is the first identified and widely investigated molluscan matrix protein and is considered 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-transfection 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 abnormal. 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 component of the NF-κB signalling pathway, Pf-Rel, can directly bind the  Nacrein  promoter in  P .  fucata , and regulate its transcription and shell formation.

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