nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that play an important role in the homeostatic regulation of physiological functions. Our previous studies showed that nAChRs in the genome of pearl oyster Pinctada fucata martensii (PmnAChRs) were expanded through tandem duplication. This study aimed to analyze the function of five tandemly duplicated PmnAChRs in the transplantation immunity in P. f. martensii. Transcriptome analysis reveals that the differentially expressed genes (DEGs) shared between PmnAChR-RNAi and the control group were functionally involved in Signal transduction, Immune system et al., and most of the related genes were down-regulated in the PmnAChR-RNAi group. The different copies of PmnAChR may regulate transplantation immunity through various pathways, such as Wnt, protein digestion and absorption, Hippo, and gap junction pathway. The inflammation factor interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were down-regulated in PmnAChR-1, 4, 5-RNAi group, and the serum from the pearl oysters in the PmnAChR-1-4-RNAi group could promote the proliferation of the Vibrio harveyi, indicating the immunosuppressive function after down-regulation of PmnAChRs. The different responses of antioxidant enzymes and diverse signal pathways after down-regulation of PmnAChRs suggested that the five tandemly duplicated PmnAChRs may cooperate with different α type PmnAChRs and constitute the functional ion channel in the membrane. Results of this study not only provide insight for the effective regulation of the transplantation immunity, but also provide a theoretical reference for the study of the adaptive evolutionary mechanism of repeating genes.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression via the recognition of their target messenger RNAs. MiR-10a-3p plays an important role in the process of ossification. In this study, we obtained the precursor sequence of miR-10a-3p in the pearl oyster Pinctada fucata martensii (Pm-miR-10a-3p) and verified its sequence by miR-RACE technology, and detected its expression level in the mantle tissues of the pearl oyster P. f. martensii . Pm-nAChRsα and Pm-NPY were identified as the potential target genes of Pm-miR-10a-3p. After the over-expression of Pm-miR-10a-3p, the target genes Pm-nAChRsα and Pm-NPY were downregulated, and the nacre microstructure became disordered. The Pm-miR-10a-3p mimic obviously inhibited the luciferase activity of the 3′ untranslated region of the Pm-NPY gene. When the interaction site was mutated, the inhibitory effect disappeared. Our results suggested that Pm-miR-10a-3p participates in nacre formation in P. f. martensii by targeting Pm-NPY. This study can expand our understanding of the mechanism of biomineralization in pearl oysters.

IntroductionIn the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene.MethodsWe constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region.ResultThe DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors.ConclusionThese findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Pre-grafting condition is an important method to promote recovery from transplant surgery during pearl production. In the present study, we constructed two DNA methylomes from pearl oysters with and without conditioning to investigate the molecular mechanism of the pearl oyster Pinctada fucata martensii underlying the pre-grafting condition. A total of 4,594,997 and 4,930,813 methyl CG in the control (Con) and pre-grafting group (PT) were detected, resulting in the whole genome methylation profile and methylation pattern in P. f. martensii. Results reveal that the promoter, especially the CpG island-rich region, was more infrequently methylated than the gene function elements in P. f. martensii. A total of 51,957 differently methylated regions (DMRs) between Con and PT were obtained, including 3789 DMR in the promoter and 16,021 in the gene body. Based on gene ontology and pathway enrichment analyses, these DMRs were mainly related to "cellular process", "metabolic process", "Epstein-Barr virus infection", and "Fanconi anemia pathway". The methylation site in the promoter region may be associated with the promoter activity and transcription factor binding. These results help our understanding of the mechanism of pre-grafting condition, thereby providing key information in guiding to improve the conditioning methods for enhanced pearl oyster survival rate after transplantation.

The miR-29 family includes miR-29a, miR-29b and miR-29c. Our previous research showed that miR-29a of Pinctada martensii (Pm-miR-29a) is involved in nacre formation via targeting neuropeptide Y receptor type 2 (Y2R). In this report, we validated and corrected the precise sequence of mature Pm-miR-29b and elucidated its function and mechanism difference with Pm-miR-29a in nacre formation. Our results showed that the precursor of Pm-miR-29b has 87 bp with a characteristic hairpin structure, which is the same with that of Pm-miR-29a. Stem-loop quantitative real-time PCR analysis indicated that Pm-miR-29b was constitutively expressed in all of the tissues, with high expression in gonads. After overexpression of Pm-miR-29b via mimic injection into P. martensii, the nacre microstructure showed a disordered growth status with a little difference with Pm-miR-29a. Target prediction analysis indicated that tyrosinase-like protein (Pfty), nacre protein (N19), fibroblast growth factor 18, protein serine kinase and neuropeptide Y2R were the potential target gene of Pm-miR-29b. Dual-luciferase analysis showed that the luciferase activity of the reporter containing 3 ' UTR of Pfty and Y2R gene was significantly inhibited by Pm-miR-29b mimics. However, only the expression of Pfty was downregulated after overexpression of Pm-miR-29b in vivo. Therefore, Pm-miR-29b participated in nacre formation via targeting Pfty in P. martensii.

Decitabine (DAC), an inhibitor of DNA methyltransferase, is typically used to reverse DNA methylation and is considered an epigenetic modifying drug. DNA methylation is crucial to the regulation of gene expression without altering genetic information. Our previous research showed that the DNA methylation levels of many immune-related genes changed after the pre-grafting condition in pearl production. In the present study, we evaluated the DNA methylation level and analyzed transcriptome, enzyme, and antimicrobial activities after DAC treatment to evaluate the effect of DAC on DNA methylation and immune system of pearl oyster Pinctada fucata martensii. Results showed that DAC significantly decreased the level of global DNA methylation in the hemocytes of the pearl oysters. Transcriptome analysis obtained 577 differentially expressed genes (DEGs) between the control and DAC treatment group. The DEGs were mainly enriched in the following pathways: “Relaxin signaling pathway,” “Cytosolic DNA-sensing pathway,” “Platelet activation,” and “Peroxisome,” and related genes were overexpressed after DAC treatment. DAC treatment resulted in a substantial increase in the levels of serum superoxide dismutase, interleukin-17, phenol oxidase, tumor necrosis factor, and antimicrobial activity, compared with the control. These results suggested that DAC can alter DNA methylation level, activate immune-related genes, and improve the level of humoral immunity in pearl oysters, thereby increasing our understanding of the mechanism underlying DNA methylation in immune regulation.