MicroRNAs (miRNAs) are a class of non-coding RNA molecules with presumed post-transcriptional regulatory activity in various biological processes, such as development and biomineralization. Pinctada martensii is one of the main species cultured for marine pearl production in China and Japan. In our previous research, 258 pm-miRNAs had been identified by solexa deep sequencing in P. martensii, while it is far from the number of miRNAs found in other species. In this study, based on the transcriptome database of pearl sac, we identified 30 candidate pm-miRNAs by computational prediction. Among the obtained 30 pm-miRNAs, 13 pm-miRNAs were generated from the complementary strand of protein-coding mRNAs, and 17 pm-miRNAs could not be annotated using blastx and tblastn analysis. Notably, 10 of the 30 pm-miRNAs, such as pm-miR-1b, pm-miR-205b and pm-miR-375b, were homologous with the reported pm-miRNAs, respectively. To validate the existence of the identified pm-miRNAs, eight randomly selected pm-miRNAs were tested by stem loop quantitative RT-PCR analyses using 5.8S as the internal reference gene. Target prediction between the obtained pm-miRNAs and biomineralization-related genes by microTar, miRanda and RNA22 indicated pm-miR-2386 and pm-miR-13b may be the key factors in the regulation network by regulating the formation of organic matrix or the differentiation of mineralogenic cell during shell formation. Thus, this study enriched miRNA databases of pearl oyster and provided a new way to understand biomineralization.

MiR-29a is a conserved miRNA that participates in bone formation and immune response in vertebrates. miR-29a of Pinctada martensii (Pm-miR-29a) was identified in the previous research though deep sequencing. In this report, the precise sequence of mature Pm-miR-29a was validated using miRNA rapid amplification of cDNA ends (miR-RACE) technology. The precursor sequence of Pm-miR-29a was predicted to have 87 bp. Stem loop qRT-PCR analysis showed that Pm-miR-29a was easily detected in all the tissues, although expressions in the mantle and gill were low. The microstructure showed the disrupted growth of the nacre after Pm-miR-29a over-expression, which was induced by mimic injection into P. martensii. Results of the target analysis indicated that neuropeptide Y receptor type 2 (Y2R) was the potential target of Pm-miR-29a. Meanwhile, Pm-miR-29a mimics could obviously inhibit the relative luciferase activity of the reporter containing 3′ UTR (Untranslated Regions) of the Y2R gene. Furthermore, the expression of Y2R was downregulated whereas expressions of interleukin 17 (IL-17) and nuclear factor κB (NF-κB) were upregulated after Pm-miR-29a over-expression in the mantle and gill, thereby suggesting that Pm-miR-29a could activate the immune response of the pearl oyster. Results showed that Pm-miR-29a was involved in nacre formation and immune response by regulating Y2R in pearl oyster P. martensii.

Bone morphogenetic protein 7 (BMP7), also called osteogenetic protein-1, can induce bone formation. In this study, the obtained full-length cDNA of BMP7 from Pinctada martensii (Pm-BMP7) was 2972 bp, including a 5'-untranslated region (UTR) of 294 bp, an open reading fragment of 1290 bp encoding a 429 amino acid polypeptide and a 3'-UTR of 1388 bp. The deduced protein sequence of Pm-BMP7 contained a signal peptide, a pro-domain and a mature peptide. The mature peptide consisted of 135 amino acids and included a transforming growth factor β family domain with six shared cysteine residues. The protein sequence of Pm-BMP7 showed 66% identity with that from Crassostrea gigas. Two unigenes encoding Pm-BMPRI (Pm-BMP receptor I) and Pm-BMPRII were obtained from the transcriptome database of P. martensii. Tissue expression analysis demonstrated Pm-BMP7 and Pm-BMPRI were highly expressed in the mantle (shell formation related-tissue), while Pm-BMPRII was highly expressed in the foot. After inhibiting Pm-BMP7 expression using RNA interference (RNAi) technology, Pm-BMP7 mRNA was significantly down-regulated (p < 0.05) in the mantle pallium (nacre formation related-tissue) and the mantle edge (prismatic layer formation related-tissue). The microstructure, observed using a scanning electron microscope, indicated a disordered growth status in the nacre and obvious holes in the prismatic layer in the dsRNA-Pm-BMP7 injected-group. These results suggest that Pm-BMP7 plays a crucial role in the nacre and prismatic layer formation process of the shell.

Marine bivalves undergo complex development processes, such as shell morphology conversion and changes of anatomy and life habits. In this study, the transcriptomes of pearl oyster Pinctada fucata martensii and Pacific oyster Crassostrea gigas at different development stages were analyzed to determine the key molecular events related to shell formation, settlement and metamorphosis. According to the shell matrix proteome, biomineralization-related genes exhibited a consensus expression model with the critical stages of shell formation. Differential expression analysis of P. f. martensii, revealed the negative regulation and feedback of extracellular matrixs as well as growth factor pathways involved in shell formation of larvae, similar to that in C. gigas. Furthermore, neuroendocrine pathways in hormone receptors, neurotransmitters and neuropeptide receptors were involved in shell formation, settlement and metamorphosis. Our research demonstrated the main clusters of regulation elements related to shell formation, settlement and metamorphosis. The regulation of shell formation and metamorphosis could be coupled forming the neuroendocrine-biomineralization crosstalk in metamorphosis. These findings could provide new insights into the regulation in bivalve development.