Deep sequencing of ESTs from nacreous and prismatic layer producing tissues and a screen for novel shell formation-related genes in the pearl oyster

Abstract

Background: Despite its economic importance, we have a limited understanding of the molecular mechanisms underlying shell formation in pearl oysters, wherein the calcium carbonate crystals, nacre and prism, are formed in a highly controlled manner. We constructed comprehensive expressed gene profiles in the shell-forming tissues of the pearl oyster Pinctada fucata and identified novel shell formation-related genes candidates.

Principal findings: We employed the GS FLX 454 system and constructed transcriptome data sets from pallial mantle and pearl sac, which form the nacreous layer, and from the mantle edge, which forms the prismatic layer in P. fucata. We sequenced 260477 reads and obtained 29682 unique sequences. We also screened novel nacreous and prismatic gene candidates by a combined analysis of sequence and expression data sets, and identified various genes encoding lectin, protease, protease inhibitors, lysine-rich matrix protein, and secreting calcium-binding proteins. We also examined the expression of known nacreous and prismatic genes in our EST library and identified novel isoforms with tissue-specific expressions.

Conclusions: We constructed EST data sets from the nacre- and prism-producing tissues in P. fucata and found 29682 unique sequences containing novel gene candidates for nacreous and prismatic layer formation. This is the first report of deep sequencing of ESTs in the shell-forming tissues of P. fucata and our data provide a powerful tool for a comprehensive understanding of the molecular mechanisms of molluscan biomineralization.

Figure 1. Tissues used for EST analysis.

A, schematics of the shell and pearl sac of the Japanese pearl oyster Pinctada fucata. Two nacreous layer-producing tissues, pallium and pearl sac, and 1 prismatic layer-producing tissue, mantle edge, were used in this study. B, C, a pearl harvested from a pearl sac used in this study (B) and its peripheral microstructure revealed by scanning electron microscopy (C).

Figure 2. Composition and distribution of 29682 genes identified in this study.

A, annotation of genes in each tissue. Blastx algorithm with cut-off E-value of 1.0E-10 was used for annotation. B, gene ontology of genes annotated with GO terms. C, distribution of genes in 3 tissues.

Figure 3. Molecular properties of 000096.

A, complete cDNA and deduced amino acid sequences of 000096. Putative signal peptide and galactose-binding lectin domain are bold and shaded, respectively, in the amino acid sequences. B, motif structure of the protein encoded by 000096 containing a signal peptide and galactose (Gal)-binding lectin domain revealed by InterProScan. C, comparison of a putative Gal-binding lectin domain encoded by 000096 with those of lectins from pearl oyster Pteria penguin (DDBJ/EMBLE/GenBank accession number: AB037167.1), Florida lancelet Branchiostoma floridae (XP_002600399), and zebrafish Danio rerio (BX950205.10). Conserved cysteine residues are indicated by arrowheads.

Figure 4. Expression of known nacreous genes.

Abbreviations used are: PFMG1, Pinctada fucata mantle gene 1; ACCBP, amorphous calcium carbonate binding protein; CaLP, calmodulin-like protein; TPM, transcripts per million; ME, mantle edge; P, pallium; PS, pearl sac. PFMG1-2, N16.6, N16.7 and N19-2 are novel isoforms found in this study (Fig. S2).

Figure 5. Expression of known prismatic genes.

Abbreviations used are: KRMP, lysine-rich matrix protein; TPM, transcripts per million; ME, mantle edge; P, pallium; PS, pearl sac. Shematrin1-2 and shematrin2-2 are novel isoforms found in this study (Fig. S3).

Figure 6. Shell formation-related gene candidates in Pinctada fucata EST library retrieved by their homology with KRMP1 and N19.

A, homology of 006605 with lysine-rich matrix protein KRMP1 . An N-terminal signal peptide is followed by a lysine-rich basic region and then by a glycine/tyrosine-rich domain (KRMP1) or G(R/W)RR(N/Y/W) repeats (006605) at the C terminus. Boxed sequences are putative signal peptides. The expression of 006605 is highly specific to the pallium. B, homology of 000496 with a nacreous gene, N19 . The boxed sequences are putative signal peptides predicted by InterProScan. The expression of 000496 is highly specific to the pallium. An N19 isoform, N19-2, was found in this study (see Fig. 4 and Fig. S2). Abbreviations are: TPM, transcripts per million; ME, mantle edge; P, pallium; PS, pearl sac.

Figure 7. Shell formation-related gene candidates in Pinctada fucata EST library retrieved by homology with PFMG1.

A, deduced amino acid sequences of 000262, 000390, 000493, 000594, and 002138. Putative signal peptides and EF-hand calcium-binding domains are in bold and shaded, respectively. A putative transmembrane domain in 000594 is boxed. B, structural properties of the genes and their expression patterns. All proteins contain EF-hand calcium-binding domains. Proteins encoded by 000296, 000493, and 0002138 contain N-terminal signal peptides, whereas 000594 has a transmembrane domain. The expression levels of 000262, 000390, and 000594 are higher in the mantle edge than in the pallium, whereas 000493 and 002138 are higher in the pallium than in the mantle edge. A PFMG1 isoform, PFMG1-2, was found in this study (Fig. 4 and Fig. S2). Abbreviations are: TPM, transcripts per million; ME, mantle edge; P, pallium; PS, pearl sac.

Figure 8. Genes clustered with known nacreous genes in their expression patterns in different tissues.

The intensity of green color corresponds to the expression levels (TPM) of each gene in different tissues. Abbreviations are: ME, mantle edge; P, pallium; PS, pearl sac; TPM, transcripts per million. *These were also retrieved as genes specific to nacre-producing tissues in Table 2. **006605 shared homology with KRMP1 (Table 3, Fig. 6A).

Figure 9. Genes clustered with known prismatic genes in their expression patterns in different tissues.

The intensity of green color corresponds to the expression levels (TPM) of each gene in different tissues. *000010, 000002, and 000009 were identified as Pinctada fucata mantle gene 10 (PFMG10), mantle gene 4 (PFMG4), and mantle gene 5 (PFMG5), respectively (Table 3). Abbreviations used are: ME, mantle edge; P, pallium; PS, pearl sac; TPM, transcripts per million.