The paired-box 3 (
) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new
gene was identified from
(
,
) (
) by RACE-PCR (rapid-amplification of cDNA ...ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of
was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed
shared the highest (69.2%) identity with
of
. Tissue expression profile showed that
had the highest expression in mantle, a nacre-formation related tissue. The
silencing significantly inhibited the expression of
,
,
and
, genes involved in
-mediated melanin synthesis, but had no effect on
and an increase effect on
. Furthermore, the
knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (
) knockdown. These data indicated that
played an important regulating role in melanin synthesis by
pathway in
.
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.
Heterodimeric PEBP2/CBFs are key regulators in diverse biological processes, such as haematopoietic stem-cell generation, bone formation and cancers. In this work, we cloned runt-like transcriptional ...factor (designated as PmRunt) and CBF β (designated as PmCBF) gene, which comprise the heterodimeric transcriptional factor in Pinctada martensii. PmRunt was identified with an open reading frame that encodes 545 amino acids and has typical Runt domain. Phylogenetic analysis results speculated that runt-like transcriptional factors (RDs) in vertebrates and invertebrates are separated into two branches. In molluscs, PmRunt and other RDs are clustered in one of these branches. Direct interaction between PmRunt and PmCBF was evidenced by yeast two-hybrid assay results. Gene repression by RNA interference decreased the expression level of PmRunt, and subsequent observation of the inner surface of the nacre by scanning electron microscopy demonstrated disordered growth. The luciferase activities of reporters that contain promoter regions of Collagen VI-like (PmColVI) and PmNacrein were enhanced by PmRunt. Meanwhile, Pm-miR-183 apparently inhibited the relative luciferase activity of reporters containing the 3'-UTR of PmRunt. The expression level of PmRunt was repressed after Pm-miR-183 was overexpressed in the mantle tissue. Therefore, we proposed that PmRunt could be targeted by Pm-miR-183 and regulate the transcription of PmColVI and PmNacrein by increasing their transcriptional activity, thereby governing nacre 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.
Mollusk shell is one kind of potential biomaterial, but its vague mineralization mechanism hinders its further application. Mollusk shell matrix proteins are important functional components that are ...embedded in the shell, which play important roles in shell formation. The proteome of the oyster shell had been determined based on the oyster genome sequence by our group and gives the chance for further deep study in this area. The classical model of shell formation posits that the shell proteins are mantle-secreted. But, in this study, we further analyzed the shell proteome data in combination with organ transcriptome data and we found that the shell proteins may be produced by multiple organs though the mantle is still the most important organ for shell formation. To identify the transport pathways of these shell proteins not in classical model of shell formation, we conducted a shell damage experiment and we determined the shell-related gene set to identify the possible transport pathways from multiple organs to the shell formation front. We also found that there may exist a remodeling mechanism in the process of shell formation. Based on these results along with some published results, we proposed a new immature model, which will help us think about the mechanism of shell formation in a different way.
► We obtained the full length of DPT gene in Pinctada martensii. ► DPT mRNA was higher expressed in the mantle and pearl sac. ► RNA interference was used to elucidate the function of DPT gene. ► The ...obtained DPT was functional for nacre formation in P. martensii.
Dermatopontin (DPT) is identified as a major component of the shell matrix protein. However, its exact function in the shell formation remains obscure. In this study, we described the characteristic and function of DPT gene from Pinctada martensii. DPT cDNA was 797bp long, containing an open reading fragment (ORF) of 537bp encoding a polypeptide of 178 amino acids with an estimated molecular mass of 21.4kDa and theoretical isoelectric point of 5.97. The 5′ untranslated region (UTR) was 11bp and the 3′UTR was 249 with 18bp poly (A) tail. In the peptide, there was a signal sequence, six potential phosphorylation sites, one glycosylation site and eight cysteine residues. Moreover, a sequence motif (D-R-X-W/F/Y-X-F/Y/I/L/M-X1–2-C) was contained and repeated itself three times in the entire sequence. DPT mRNA was constitutively expressed in all studied tissues with the most abundant mRNA in the mantle, which was nacre formation-related tissue. After decreasing DPT expression using RNA interference (RNAi) technology in the mantle, the nacreous layer showed a disordered growth; whereas the prismatic layer of the shells has no significant changes. These results suggested that DPT obtained in this study was a constitutive matrix protein and participated in nacre formation in P. martensii.
MicroRNAs (miRNAs) are noncoding RNA molecules that function as negative regulators of target genes. In our previous research, 258 pm-miRNAs were identified in Pinctada martensii by Solexa deep ...sequencing. Pm-miR-2305 was one of the identified pm-miRNAs with a potential function in biomineralization. In the present study, the precursor of pm-miR-2305 was predicted with 96 bp, containing a characteristic hairpin structure. Stem-loop qRT-PCR analysis indicated that pm-miR-2305 was constitutively expressed in all the tissues (adductor muscle, gill, mantle, hepatopancreas, foot, and gonad) of P. martensii and was highly expressed in the foot. After the over-expression of pm-miR-2305 in the mantle by mimics injection into the muscle of P. martensii, nacre demonstrated disorderly growth, as detected by scanning electron microscopy. Dual luciferase reporter gene assay indicated that pm-miR-2305 mimics could significantly inhibit the luciferase activity of the reporter containing the 3'UTR of the pearlin gene. Western blot analysis demonstrated that the protein expression of pearlin was down-regulated in the mantle tissue after the over-expression of pm-miR-2305. Therefore, our data showed that pm-miR-2305 participated in nacre formation by targeting pearlin in P. martensii.
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.
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.
Tyrosinase is a type-3 copper protein with six conserved histidine residues within the copper-binding sites. It participates in mollusk nacre formation. Here, we identified nacreous-layer-specific ...tyrosinases (NLSTyr) from
Pinctada fucata martensii
(
PmTyr-4
and
PmTyr-6
), as well as their homologs in
Pinctada maxima
(
PmaxTyr
and
PmaxTyr4
) and
Pinctada margaritifera
(
PmarTyr
and
PmarTyr-4
), which encoded tyrosinases without the six conserved histidine residues within the copper-binding sites.
PmTyr-4
and
PmTyr-6
mRNAs were spatially concentrated in the mantle central and pearl sac, which are the organs responsible for nacre formation. During shell regeneration and pearl formation,
PmTyr-4
and
PmTyr-6
were also significantly highly expressed in the mantle and pearl sac. RNA interference showed that
PmTyr-4
participated in nacreous-layer formation. The recombinant protein of PmTyr-4 (rPmTyr-4) inhibited the calcium carbonate precipitation rate. Correspondingly, calcium carbonate crystallization assay showed that the aragonite crystals of the rPmTyr-4 group were smaller than those of the control group. Moreover, the calcite and aragonite morphologies of the rPmTyr-4 group were modified compared with the control group. These results suggested that NLSTyr in pearl oyster inhibited calcium carbonate precipitation and affected crystal morphologies during nacre formation. Our findings provided new insights into the evolution and function gain of tyrosinase in Mollusk.
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