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Shell matrix proteins have important roles in the biomineralization of shells. In this study, we isolated and identified a novel shell matrix protein gene, hic7, from the mussel Hyriopsis cumingii. The cDNA of hic7 was 459 bp long, including a 240-bp open reading frame. It encoded a 79 amino acid-long protein, with amino acids 1-19 constituting the signal peptide. The resulting hic7 is rich in cysteine (16.5%). After removing the signal peptide, the molecular weight was 8.85 kDa and the theoretical isoelectric point was 6.34, indicating that hic7 is a weakly acidic shell matrix protein. Hic7 is mainly expressed in the mantle tissue of H. cumingii. In situ hybridization showed hic7 signals at the edge and dorsal region of the mantle outer fold, indicating that it is related to the formation of the prismatic and nacreous layer of the shell. RNA interference indicated that when hic7 was inhibited by 80%, the crystal morphology of the prism and nacre layers of the shell were irregular and disordered. In addition, the expression of hic7 during the early development of the pearl sac indicated that it has an important role in the transformation of calcium carbonate crystals from a disordered to an orderly deposition pattern. These results suggest that matrix protein hic7 take part in constructing the framework of crystal nucleation and regulating the calcium carbonate crystal morphology of the nacreous and prismatic layers of shells and pearls., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus which hold the potential function in the shell formation as shell matrix proteins. In this study, a C1qDC protein was identified and characterized in pearl oyster (Pinctada fucata martensii) (PmC1qDC) to explore its function in nacre formation. The PmC1qDC-deduced protein sequence carried a typical globular C1q (gC1q) domain that possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1qDC family members and shared high homology with other gC1q domains. Homologous analysis of PmC1qDC presented it contained conserved secondary structure and Phe135, Phe155, Tyr166, Phe173, Tyr181, Phe183, and Phe256 amino acid residues. Expression pattern analysis showed that PmC1qDC expressed in all the detected tissues and exhibited a significantly higher expression level in nacre formation-associated tissues. After the shell notching, the expression level of PmC1qDC showed significantly up-regulation after 12 h in the central zone of mantle (MC). PmC1qDC expression significantly decreased in the MC after RNA interference (RNAi). Furthermore, disordered crystals with evident rough surface and irregular crystal tablets were observed in the nacre after RNAi. Results suggested that PmC1qDC affects the shell nacre formation, which is significant to improve the pearl production of pearl oyster., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Chitin participates in shell formation as the main component of an organic framework. Chitin-binding protein contains domains that can bind to chitin specifically. In this study, a novel chitin-binding protein from Pinctada fucata martensii (PmCBP) with poly (chitin-binding domain) was cloned, which contains a 5'-untranslated region (UTR) of 114 bp and 3'UTR of 116 bp, and encodes a putative protein of 2044 amino acids. The predicted PmCBP protein was structurally typical of the CBP family with 20 ChtBD2 domains. Phylogenetic and linear relation analyses showed that the ChtBD2 domain has a highly conserved structure among the three species of P. f. martensii, Crassostrea gigas, and Mizuhopecten yessoensis. qRT-PCR and in-situ hybridization analysis revealed that PmCBP was most abundant in the mantle pallium whose expression level was significantly correlated with the growth traits. After RNAi, PmCBP expression was significantly inhibited in the mantle pallium (P < 0.05) and the microstructure of nacreous layers showed a disordered growth in the experiment group. These results indicated that PmCBP may be involved in nacreous layer formation through participation in the process of binding chitin in pearl oyster P. f. martensii., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mantle tissue from the black-lip pearl oyster, Pinctada margaritifera, was cultured in vitro using sterilized seawater supplemented with 0.1% yeast extract as the culture medium. Granular and agranular epithelial cells, hyalinocytes, and fibroblast-like cells were observed in the initial stages of culture. Epithelial cells later formed pseudopodial cell networks containing clusters of granulated cells, which upon maturation released their colored granules. These granules induced formation of nacre crystal deposits on the bottom of the culture plate. Cultures comprised of only granulated epithelial cells were established through periodic sub-culturing of mantle cells and maintained for over 18 mo in a viable condition. Reverse transcriptase PCR of cultured cells demonstrated gene expression of the shell matrix protein, nacrein. To further evaluate the functional ability of cultured granulated epithelial cells, nuclear shell beads were incubated in culture medium containing these cells to induce nacre formation on the beads. Observation of the bead surface under a stereomicroscope at periodic intervals showed the gradual formation of blackish yellow colored nacre deposits. Examination of the bead surface by scanning electron microscopy and energy dispersive X-ray analysis at periodic intervals revealed a distinct brick and mortar formation characteristic of nacre, comprised of aragonite platelets and matrix proteins. Calcium, carbon, and oxygen were the major elements in all stages examined. Our study shows that mantle epithelial cells in culture retain the ability to secrete nacre and can therefore form the basis for future studies on the biomineralization process and its application in development of sustainable pearl culture.

Nucleated pearls are produced by molluscs of the Pinctada genus through the biomineralisation activity of the pearl sac tissue within the recipient oyster. The pearl sac originates from graft tissue taken from the donor oyster mantle and its functioning is crucial in determining key factors that impact pearl quality surface characteristics. The specific role of related gene regulation during gem biogenesis was unknown, so we analysed the expression profiles of eight genes encoding nacreous (PIF, MSI60, PERL1) or prismatic (SHEM5, PRISM, ASP, SHEM9) shell matrix proteins or both (CALC1) in the pearl sac (N = 211) of Pinctada margaritifera during pearl biogenesis. The pearls and pearl sacs analysed were from a uniform experimental graft with sequential harvests at 3, 6 and 9 months post-grafting. Quality traits of the corresponding pearls were recorded: surface defects, surface deposits and overall quality grade. Results showed that (1) the first 3 months of culture seem crucial for pearl quality surface determination and (2) all the genes (SHEM5, PRISM, ASP, SHEM9) encoding proteins related to calcite layer formation were over-expressed in the pearl sacs that produced low pearl surface quality. Multivariate regression tree building clearly identified three genes implicated in pearl surface quality, SHEM9, ASP and PIF. SHEM9 and ASP were clearly implicated in low pearl quality, whereas PIF was implicated in high quality. Results could be used as biomarkers for genetic improvement of P. margaritifera pearl quality and constitute a novel perspective to understanding the molecular mechanism of pearl formation.

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.