Tyrosinase （Ty） is a common enzyme found in many different animal groups. In our previous study, genome sequencing revealed that the Ty family is expanded in the Pacific oyster （Crassostrea gigas）. ...Here, we examine the larger number of Ty family members in the Pacific oyster by high-level structure prediction to obtain more information about their function and evolution, especially the unknown role in biomineralization. We verified 12 Ty gene sequences from Crassostrea gigas genome and Pinctadafucata martensii transcriptome. By using phylogenetic analysis of these Tys with functionally known Tys from other molluscan species, eight subgroups were identified （CgTy_sl, CgTy s2, MolTy sl, MolTy-s2, MolTy-s3, PinTy-s 1, PinTy-s2 and PviTy）. Structural data and surface pockets of the dinuclear copper center in the eight subgroups of molluscan Ty were obtained using the latest versions of prediction online servers. Structural comparison with other Ty proteins from the protein databank revealed functionally important residues （HA1, HA2, HA3, HB1, HB2, HB3, Z l-Z9） and their location within these protein structures. The structural and chemical features of these pockets which may related to the substrate binding showed considerable variability among mollusks, which undoubtedly defines Ty substrate binding. Finally, we discuss the potential driving forces of Ty family evolution in mollusks. Based on these observations, we conclude that the Ty family has rapidly evolved as a consequence of substrate adaptation in mollusks.
Pearl oyster Pinctada fucata martensii is cultured for producing round nucleated pearls. Pearl production involves a surgical operation where a mantle tissue graft from a donor oyster and a round ...nucleus are implanted in the gonad of a host oyster. Whether the mantle graft implanted in the gonad of a host oyster contributes to the formation of a pearl sac that secretes pearl nacre to form a pearl should be determined. In April 2012, two full-sib families were separately used as donor and host oysters for a nucleus insertion operation. The pearl sac was sampled from the host oysters at day 60 after nucleus operation. A large number of simple sequence repeat (SSR) markers were developed using Illumina HiSeq™ 2000 platform. The two full-sib families were also used to mine diagnostic SSR markers for genotyping donor oyster, host oyster, and pearl sac.
A total of 3168 microsatellite loci were identified in 39,078 unigenes, and 1977 SSR primers were designed by Primer 3.0. Forty-seven SSR primers were validated, and the rate of successful amplification was 72.3%. Two diagnostic SSR primers could successfully genotype pearl sac, donor oyster, and host oyster. Donor and host oysters were both homogenous, and the alleles in pearl sac were identical to those in donor and host oysters.
The present results confirmed that the mantle graft implanted in the gonad of host oyster contributed to the formation of the pearl sac in pearl oyster P. fucata martensii.
C4ST-1 catalyzes the transfer of sulfate groups in the sulfonation of chondroitin during chondroitin sulfate synthesis. Chondroitin sulfate consists of numerous copies of negatively charged sulfonic ...acid groups that participate in the nucleation process of biomineralization. In the present study, we obtained two CHST11 genes (PmCHST11a and PmCHST11b) which encoded the C4ST-1 and explored the functions of these genes in the synthesis of chondroitin sulfate and in the formation of the nacreous layer of shells.
Both PmCHST11a and PmCHST11b had a sulfotransferase-2 domain, a signal peptide and a transmembrane domain. These properties indicated that these genes localize in the Golgi apparatus. Real-time PCR revealed that both PmCHST11a and PmCHST11b were highly expressed in the central zone of the mantle tissue. Inhibiting PmCHST11a and PmCHST11b via RNA interference significantly decreased the expression levels of these genes in the central zone of the mantle tissue and the concentration of chondroitin sulfate in extrapallial fluid. Moreover, shell nacre crystallized irregularly with a rough surface after RNA interference.
This study indicated that PmCHST11a and PmCHST11b are involved in the nacre formation of Pinctada fucata martensii through participating in the synthesis of chondroitin sulfate.
The immune response after allograft or xenograft transplantation in the pearl oyster is a major factor that cause its nucleus rejection and death. To determine the mechanism underlying the immune ...response after allograft and xenograft transplantations in the pearl oyster Pinctada fucata martensii, we constructed two sets of transcriptomes of hemocytes at different times (6 and 12 h; 1, 3, 6, 12, and 30 d) after allograft and xenograft transplantations, in which the xenografted mantle tissue was from Pinctada maxima. The transcriptomic analysis reveals many genes are involved in the immune response to transplantation, such as transient receptor potential cation channel (TRP), calmodulin (CaM), DNA replication-related genes, and sugar and lipid metabolism-related genes. The expression of these identified genes was higher in the host pearl oyster transplanted with xenograft than that by allograft. The histological analysis of the pearl sac also confirmed that many hemocytes were still gathered around the transplanted nucleus, and no pearl sac was formed in the host pearl oysters at 30 d after xenograft transplantation. The genomic analysis indicated that pearl oysters evolved many copies of genes, such as TRP, CaM, and GST, to sense and cope with the immune response after transplantation. “Ribosome” and “Cytosolic DNA-sensing pathway” were specifically induced in the xenograft group, whereas “Notch signaling pathway” specifically responded to the allograft transplantation. These results can improve our understanding of the mechanism underlying the immune response of pearl oysters after allograft and xenograft transplantations.
•Fifteen transcriptomes of the hemocytes from pearl oyster were constructed.•Obtained many pathways and genes involved in transplantation response.•Find out the difference in molecular response between allograft and xenograft.•Pearl oysters evolved many copies of genes involved in transplantation response.
•Shell injury induced the production of serum ecdysone to regulate shell repair.•The ecdysone signal was transmitted by PmEcR ecdysone receptor in pearl oyster.•Ecdysone induced the expression of ...biomineralization related genes.•PmEcR and PmRXR was located in the nuclei and cytoplasm.•PmEcR and PmRXR did not form dimers which is different from other results.
Ecdysone exists in arthropods, Mollusca and other invertebrates and plays vital roles in exoskeleton formation of Ecdysozoa. However, little is known about its functions in bivalve species. Herein, we identified ecdysone from the serum of pearl oyster Pinctada fucata martensii and obtained the coding sequence of ecdysone receptor (PmEcR) and homologue of its heterodimer protein retinoid X receptor (PmRXR). The deduced amino acid sequences of PmEcR and PmRXR contained a DNA-binding and ligand-binding domain and were very similar to the orthologs of other species. Moreover, PmEcR and PmRXR were located in the nuclei and cytoplasm of HEK-293T cells. PmEcR and PmRXR were highly expressed in early embryos and biomineralized mantle tissue. Moreover, the serum concentration of ecdysone significantly increased at 2, 4, 6, and 8 h post-shell notching. The expression of PmEcR in the mantle tissue was significantly induced at the corresponding time points, while that of PmRXR was significantly induced at 6 h. Ecdysone stimulation remarkably induced the expression of growth factors (BMP2 and BMP7), transcription factors (PmRunt and AP-1), and shell matrix protein genes (chitinase, lysine-rich matrix protein (KRMP), TYR2, and PmCOLVI), which indicated that ecdysone signaling plays important roles in shell repair. However, yeast two-hybrid assay and bimolecular fluorescence complementation showed that PmEcR and PmRXR did not form dimers, suggesting the different molecular interactions of EcR in bivalves. These findings provide insights into the function of ecdysone and its regulation pathway in bivalve species.
Long non-coding RNAs (LncRNAs) are abundant in the genome of higher forms of eukaryotes and implicated in regulating the diversity of biological processes partly because they host microRNAs (miRNAs), ...which are repressors of target gene expression. In vertebrates, miR-133 regulates the differentiation and proliferation of cardiac and skeletal muscles. Pinctada martensii miR-133 (pm-miR-133) was identified in our previous research through Solexa deep sequencing. In the present study, the precise sequence of mature pm-miR-133 was validated through miR-RACE. Stem loop qRT-PCR analysis demonstrated that mature pm-miR-133 was constitutively expressed in the adductor muscle, gonad, hepatopancreas, mantle, foot, and gill of P. martensii. Among these tissues, the adductor muscle exhibited the highest pm-miR-133 expression. Target analysis indicated that pm-RhoA was the potential regulatory target of pm-miR-133. Bioinformatics analyses revealed that a potential LncRNA (designated as Lnc133) with a mature pm-miR-133 could generate a hairpin structure that was highly homologous to that of Lottia gigantea. Lnc133 was also highly expressed in the adductor muscle, gill, hepatopancreas, and gonad. Phylogenetic analysis further showed that the miR-133s derived from chordate and achordate were separated into two classes. Therefore, Lnc133 hosting pm-miR-133 could be involved in regulating the cell proliferation of adductor muscles by targeting pm-RhoA.
•The sequence of pm-miR-133 was validated with the most abundant in adductor muscle.•Pm-RhoA was the potential regulatory target of pm-miR-133.•One potential LncRNA (designed as Lnc133) could be the primary RNA of pm-miR-133.
Color polymorphisms in molluscan shells play an important economic in the aquaculture industry. Among bivalves, shell color diversity can reflect properties such as growth rate and tolerance. In ...pearl oysters, the nacre color of the donor is closely related to the pearl color. Numerous genes and proteins involved in nacre color formation have been identified within the exosomes of the mantle. In this study, we analyzed the carotenoids present in the mantle of gold- and silver-lipped pearl oysters, identifying capsanthin and xanthophyll as crucial pigments contributing to coloration. Transcriptome analysis of the mantle revealed several differentially expressed genes (DEGs) involved in color formation, including ferric-chelate reductase, mantle genes, and larval shell matrix proteins. We also isolated and identified exosomes from the mantles of both gold- and silver-lipped strains of the pearl oyster Pinctada fucata martensii, revealing the extracellular transition mechanism of coloration-related proteins. From these exosomes, we obtained a total of 1223 proteins, with 126 differentially expressed proteins (DEPs) identified. These proteins include those associated with carotenoid metabolism and Fe(III) metabolism, such as apolipoproteins, scavenger receptor proteins, β,β-carotene-15,15'-dioxygenase, ferritin, and ferritin heavy chains. This study may provide a new perspective on the nacre color formation process and the pathways involved in deposition within the pearl oyster P. f. martensii.
The oyster species Pinctada maxima is cultured for the production of large pearls with high economic value. Pearl weight and thickness are related to the growth of P. maxima. The molecular mechanism ...underlying the growth of this species, however, remains poorly understood given the limited availability of the genetic and genomic information of this species. Here, the molecular mechanism of the asynchronous growth of P. maxima was investigated. The transcriptomes of large and small P. maxima individuals were sequenced using the Illumina HiSeq 2000 platform. A total of 145,877 unigenes were generated for the transcriptomes, and 1,921 differentially expressed genes (DEGs) were identified. Compared with the slow-growing group, the fast-growing group showed 879 and 1,042 significantly up- and down-regulated DEGs, respectively. The differential expression patterns of nine selected genes were obtained through real-time quantitative polymerase chain reaction analysis and showed consistency with those obtained through RNA-Seq analysis. The results of this study provide further insight on the complexity of the differential growth patterns of P. maxima individuals and will help guide the design of breeding programs for this economically important species.
•The basic knowledge on molecular mechanisms of unsynchronized growth of Pinctada maxima was studied.•Gene expression patterns of Pinctada maxima with different growth performances were different.•Many differentially expressed genes were linked to growth and immune responses.
Keratan sulfate possesses considerable amounts of negatively charged sulfonic acid groups and participates in biomineralization. In the present study, we investigated characteristics and functions of ...a CHST1 gene identified from the pearl oyster Pinctada fucata martensii (PmCHST1b) which participated in the synthesis of keratan sulfate. PmCHST1b amino acid sequence carried a typical sulfotransferase-3 domain (sulfotransfer-3 domain) and belonged to membrane-associated sulfotransferases. Homologous analysis of CHST1 from different species showed the conserved motif (5′ PSB motif and 3′ PB motif) which interacted with 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Structure analysis of sulfotransferase domain indicted that PmCHST1b showed the conserved catalytic structure character and the relationships presented in the phylogenetic tree conformed to that of traditional taxonomy. Expression pattern of PmCHST1b in different tissues and development stages showed that PmCHST1b widely expressed in all the detected tissues and development stages and showed the highest expression level in the central zone of mantle (MC). PmCHST1b expressed highly in the trochophore, D-stage larvae and spat which corresponded to prodissoconch and dissoconch shell formation, respectively. RNA interference (RNAi) successfully inhibited expression level of PmCHST1b in MC (P<0.05), and sulfate polymer content in the extrapallial fluid significantly reduced (P<0.05). Crystallization of shell nacre became irregular. Results above indicated that PmCHST1b may affect nacre formation by participating in synthesis of keratan sulfate in extrapallial fluid. This study provided fundamental materials for further research on the role of sulfotransferases and keratan sulfate in nacre formation.
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