| Hyriopsis cumingii is known as the main freshwater pearl-producing mussel in China,which has a great value of economic.Mantle implantation is vital for pearl culture,the sabio from donor mussels was transplanted into the mantle of recipient mussels,proliferating and degenerating to a thin outer epithelial cell layer to form a pearl-sac.Subsequently,the pearls are generated inside the pearl sacs.However,the operation poses a tremendous stress to host mussels,resulting in the nucleus discharge and increasing the risk of bacterial infection that can even lead to death.Hence,it is urgent to expand our knowledge about the physiological changes of recipient mussels and donor mantle pieces to reveal specific immune molecule mechanisms against transplant adaptation.These findings help guide us take novel management strategies of development at the key time points during the pearl sac formation and pave the way for pearling industry.In the present study,we collected the pearl sacs at different time points: 0h,2 h,6 h,12 h,24 h,48 h,96 h,7 days,14 days,and 30 days to conduct the transcriptome library of H.cumingii in the pearl sac post implantation using Next Generation Sequencing technology.The transcriptome sequencing yielded a total of 293863 unigenes with a mean length of 588 bp and N50 length of 840 bp,and 27176 assembled unigenes were matched to homogeneous sequences in at least one database.When we mapped all the sequences to pathways in the Kyoto Encyclopedia of Genes and Genomes(KEGG)database,it was found that a variety of genes were involved in immune signalingpathways such as Toll-like receptor signaling pathway?Complement and coagulation cascades?NOD-like receptor signaling pathway?B cell receptor signaling pathway?Chemokine signaling pathway.Next,we performed pairwise comparisons of all the samples with a strict distribution algorithm and identify 4878 significant differentially expressed genes(DEGs).A principle component analysis and the heat map clustering were performed based on the expression profiles of DEGs.According to the analysis results,we speculated that the physical condition of the recipient mussels and donor mantle pieces returned to normalafter implantation for about one month,which was divided into six vital phases(0,2 to 6 h,12 to 24 h,48 h to 7 days,14 days,and 30 days)on the basis of the overall similarities in the DEGs among the samples.We compared the DEGs between 0/2 h,6 h/12 h,24 h/48 h,7 days/14 days,and 14 days/30 days to gain an insight of the dynamic physiological changes that occur in mussels during pearl sac formation.We further performed GO and KEGG pathway enrichment analyses of the five groups to investigate the molecular mechanisms and capture the key genes in Complement and coagulation cascades,MAPK signaling pathway,TLR signaling pathways and Chitin metabolic process during pear sac formation.Additionally,eight DEGs were selected for the qRT-PCR analysis and the significant correlation coefficient(r)values of the expression patterns determined with qRT-PCR and RNA-seq ranged from 0.86 to 0.97.The results demonstrated that the expression patterns of the selected DEGs determined by the two methods were identical and the transcriptomic profiles were accurate and reliable.We emphatic researched the Interleukin-1 receptor-associated kinase 4(IRAK4)and Tumor necrosis factor receptor-associated factor 6(TRAF6)in the Toll-like receptor signaling pathway via cell biology and molecular biology methods.We cloned an IRAK4 homolog from H.cumingii,the cDNA sequence of HcIRAK4 was 2595 bp in length.The open reading frame was 1674 bp,encoding a putative polypeptide of 557 amino acid residues,with a predicted molecular weight of approximately 62.3 kDa and a theoretical isoelectric point(PI)of 5.34.The protein of HcIRAK4 is highly observedand contains an N-terminal death domain(DD,residues 5–115)and the typical serine/threonine/tyrosine protein kinase domain(STYKc,residues 270–553).qRT-PCR analysis showed that the HcIRAK4 mRNA was expressed ubiquitously in all candidate tissues and abundantly expressed in hemocytes,gills,and foot.The up-regulation of HcIRAK4 transcription after immune challenge with lipopolysaccharides(LPS)and live Aeromonas hydrophila suggested its function in innate immunity against invasive pathogen colonization.Meanwhile,the expression pattern of HcIRAK4 has the high similarity with HcMyD88,indicating that TLR-mediated pathway participates in the immune responses at the early stage of pathogen invasion.The subcellular localization of HcIRAK4 showed its distribution in the cytoplasm of HEK293 T cells.Overexpression of HcIRAK4 significantly repressed the transcriptional activity of NF-?B and AP-1 induced by HcMyD88,while no obvious changes occurred when the cells were transfected with HcIRAK4;we inferred that a negative feedback regulatory mechanism of IRAK4 in TLR signaling in lower animals may exist.The complete cDNA sequence of HcTRAF6 from H.cumingii was 3986 bp long,and the 1965-bp open reading frame encodes a predicted product protein of 654 amino acid residues,including an N-terminal RING domain ? two TRAF-type zinc finger domains?a typical coiled coil and a meprin and TRAF-C-terminal(MATH)domain.HcTRAF6 was transcribed in all examined tissues of H.cumingii,with enrichment in gills.The transcription of HcTRAF6 was obviously induced in the immune tissues?gills and hemocytes after immune stimulation,showing that HcTRAF6 can respond to the bacterial infection positively.The dual-luciferase reporter assay presented that the over-expression HcTRAF6 had the capacity of enhancing the activity of NF-?B reporter in a dose-dependent manner.The RNA interference strategy was applied for further investigation of HcTRAF6 physiological function.The up-regulation of antimicrobial peptides in anti-bacterial infection was strongly suppressed in the HcTRAF6-silenced mussels and the depleted HcTRAF inhibited the elimination of A.hydrophila. |
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