Effect of Pf-Rel/poI-?B RNA interference on gene expression in mantle tissue in Pinctada fucata

Pinctada fucata is an important pearl production shellfish in aquaculture. The formation of shells and pearls is a hot research topic in biomineralization, and matrix proteins secreted by the mantle tissues play the key role in this process. However, upstream regulatory mechanisms of transcription factors on the matrix protein genes remain unclear. Previous studies have shown that NF-?B signaling pathway regulated biomineralization process through expression regulation of specific matrix proteins.In this study, we systematically investigated the effect of the NF-?B signaling pathway key factor Pf-Rel and inhibitory protein poI-?B on the biomineralization and shell regeneration process. RNA-seq was conducted based on RNA interference animal model to identify potential regulatory genes by transcription factor Pf-Rel. We extracted total RNA from the whole tissue samples of the pericardium 7 days after dsRNA injection, and named them as the Rel-RNAi group, I?B-RNAi group and control group, respectively. We first calculated the Pearson correlation coefficient between each pair of samples, which reflects the similarity in overall gene expression patterns among the samples. The calculation results showed a high correlation coefficient of 0.954 between the Rel-RNAi group and I?B-RNAi group. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on differentially expressed genes (DEGs) from two groups. The top three enriched GO terms for DEGs were: extracellular region, peptidase inhibitor activity and peptidase regulator activity. In addition, immune receptor activity and integrin-mediated signaling pathways terms were significantly enriched. The KEGG Pathway classification showed that a large number of DEGs were classified as signal transduction and immune system terms. Biomineralization is also an important natural immune pathway in shellfish, which suggests that the NF-?B signaling pathway in P. fucata is closely related to natural immunity. KEGG pathway enrichment analysis indicated that substantial DEGs were attributed to PI3K-Akt signaling pathway term, which plays a vital role in cell survival Overall design: To investigate the regulatory effcet of NF-?B signaling pathway on biomineralization, we established RNA interference animial model in pearl oysters, P. fucata. Healthy adult oysters with similar size and weight were injected with 30 µg different dsRNAs into the adductor muscle, and each treatment group included five oysters. Control group was injected with 30 µg gfp dsRNA. After 7 days, the mantle tissues from five individuals were collected for total RNA extraction and RNA-seq.