Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many aspects of the biomineralization process and its evolutionary origin remain unknown. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms that underlie its production of shells and pearls are not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins, chondroitin sulfotransferases, and regulatory elements. Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre suggests that elements of chitin- and collagen-based matrices have deep roots and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems.

Pearl oyster Pinctada martensii is cultured for production of pearl in China. It needs to implant a mantle graft cut from a donor oyster and a seed nucleus into the gonad of the host oyster to produce a pearl. Pearl sac surrounding the nucleus is formed by the proliferation of the implanted mantle graft from the outer mantle epithelial cells in the host oyster. The pearl sac is responsible for production of a cultured pearl. A comprehensive transcriptome analysis on pearl sac will help to understand the mechanism on pearl formation and immune response of host oyster after nucleus implantation. In the present study, 39,400,004 reads were produced from the pearl sac using RNA-sequence technology and then assembled into 102,762 unigenes. More than 22.4% of these unigenes were possibly involved in approximately 219 known signaling pathways. A total of 37,188 unigenes were annotated based on sequences similarities with known proteins. Fifty-one biomineralization-related unigenes and 268 immune-related unigenes were not previously detected in P. martensii. The un-annotated unigenes may be some genes specifically existed in P. martensii. These annotated or un-annotated unigenes in the present studies were valuable for the future investigation on molecular mechanism of pearl formation and immune response of the species.

MicroRNAs (miRNAs) are short-nucleotide RNA molecules that function as negative regulators of gene expression in various organisms. However, miRNAs of Pinctada martensii have not been reported yet. P. martensii is one of the main species cultured for marine pearl production in China and Japan. In order to obtain the repertoire of miRNAs in P. martensii, we constructed and sequenced small RNA libraries prepared from P. martensii by Solexa deep sequencing technology and got a total of 27,479,838 reads representing 3,176,630 distinct sequences. After removing tRNAs, rRNAs, snRNAs, and snoRNAs, 10,596,306 miRNA reads representing 18,050 distinct miRNA reads were obtained. Based on sequence similarity and hairpin structure prediction, 258 P. martensii miRNAs (pm-miRNA) were identified. Among these pm-miRNAs, 205 were conserved across the species, whereas 53 were specific for P. martensii. The 3' end sequence of U6 snRNA was obtained from P. martensii by 3' rapid amplification of cDNA end PCR reaction and sequence-directed cloning. Eight conserved pm-miRNAs and two novel pm-miRNAs were validated by stem-loop quantitative real-time PCR with U6 snRNA as an internal reference gene. pm-miRNAs and the reported biomineralization-related genes were subjected to target analysis by using target prediction tools. Some of the pm-miRNAs, such as miR-2305 and miR-0046, were predicted to participate in biomineralization by regulating the biomineralization-related genes. Thus, this study demonstrated a large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation.

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