Effects And Its Mechanism Of Ca²⁺Concentration On The Deposition Of Nacre In Hyriopsis Cumingii

Calcium availability is identified as one of the major limiting factors on the growth of mollusks and pearl quality. However, the molecular mechanism of calcium concentration affecting on the nacre deposition in shell remains unknown. In present study, the Ca2+is treated as a crucial environmental factor to screen the suitable Ca2+concentration for nacre deposition of the freshwater pearl mussel Hyriopsis cumingii by measuring the nacre deposition, nacre morphology, and activities of the Ca2+metabolism related enzymes. The results will provide basic data for pearl culture industry. Digital gene expression profiling analyses (DGE) is performed to screen the differential expression genes which participate in nacre deposition, and to confirm the main metabolism pathways and signal pathways which Ca2+affects on nacre deposition. These results will hope to illuminate the molecular mechanism of Ca2+affecting on nacre deposition, and will provide new research approach for molluscan biomeniralization.Two novel cDNA-coding genes, CaM and CaLP were cloned and characterized from the freshwater pearl mussel H. cumingii by RACE technology with full-length sequences of726bp and1217bp, respectively. Tissue-specific expression analysis revealed that CaM mRNA was prominent expressed in the gill, mantle center and foot. In contrast, CaLP mRNA was mainly expressed in the mantle edge. The result of Ca2+dependent electrophoretic shift showed that recombinant CaLP has more Ca2+-binding affinity than recombinant CaM. The calcium stimulation test lasted5weeks implied that CaM and CaLP had differential expression patterns in response to various environmental Ca2+concentrations (0.25-1.25mM). The expression of CaM mRNA was up-regulated by low Ca2+concentration (0.25mM), and the highest expression of CaLP mRNA occurred under Ca2+concentration of1.0mM.An a-CA (HcCA) were cloned and characterized from H. cumingii by RACE technology. The full length of HcCA cDNA is1911bp which encodes550aa. The spatial and temporal patterns of expression of HcCA indicate that this gene mainly expressed in the mantle of juvenile mussels. This result suggests that HcCA may play an important role in shell growth. The expression profile of HcCA under various environmental conditions, including Ca2+concentration, air exposure, pH and water temperature reveal that the transcription of HcCA is significantly affected by external environmental conditions. Our results suggest that HcCA play important roles in calcium absorption and pH homeostasis in mantle.In the culture experiment of10weeks under eight (0.25-2.0mM) Ca2+concentrations, the growth traits and nacre deposition in2years old mussels were higher at the Ca2+concentrations of1.0-1.75mM than those at the other Ca2+concentrations. The results of Raman spectra and Scanning electron microscopy analyses implied that the nacre sheet at the Ca2+concentration of1.5mM contained more shell matrix than the other sheet at the Ca2+concentration of0.25mM or2.0mM. Meanwhile, the results of enzymic activities of alkaline phosphatase, Ca2+-ATPase, and Mg2+-ATPase in the tissues of gill and mantle pallial suggested that the Ca2+concentrations of1.0-1.75mM is benefit for Ca2+absorption and accumulation in-mussels. We conclude that the Ca2+concentrations of1.0-1.75mM are suitable for the growth and nacre deposition of2years old mussels.Using RNA-seq technologies on an Illumina HiSeq2000platform, we assembled a de novo transcriptome of mantle pallial in H. cumingii, produced50million high-quality reads, and assembled into61,119Unigenes with an averge length of450bp. Blastp were performed to search the the deduced protein sequences of Unigenes in the Genbank non redundant protein database (NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO) databases respectively, and total15,655Unigenes were annotated. Among them,6,322,11,670, and6,322Unigenes were categorized into55GO terms,25COG categories, and257KEGG pathways. Meanwhile,37of Unigenes encoded shell matrix proteins,55of Unigenes encoded biomineralization-related proteins, and94of Unigenes encoded calcium metabolism related proteins were founded.Digital gene expression profiling analyses based on RNA-Seq were performed to test for differential expression across the treatment groups at the Ca2+concentration of 0.25mM,1.5mM and2.0mM. The results showed that there were1604up-regulated DEGs and1489down-regulated DEGs between groups of1.5mM and0.25mM; and762up-regulated DEGs and596down-regulated DEGs in between groups of1.5mM and2.0mM. Among the DEGs,4DEGs encode shell matrix proteins including Papilin, Shematrin8, Nacre serine protease inhibitor1, and Perlucin6encode,13DEGs encode biomineralization-related proteins such as Chitin synthase, Carbonic anhydrase, C-type lectin, and5DEGs encode calcium metabolism related proteins, such as Calmodulin-like protein, Sarcoplasmic calcium-binding protein, Troponin C. KEGG pathway analyses showed that total384DEGs were riched into24pathways including Vitamin digestion and absorption, Protein digestion and absorption, Phagosome, and Tyrosine metabolism between groups of1.5mM and0.25mM; while426DEGs were riched into35pathways including Vitamin digestion and absorption, Focal adhesion, Phagosome, Regulation of actin cytoskeleton between groups of1.5mM and2.0mM. These results suggested that the DEGs and the riched pathways mentioned above may be the main genes and pathways that Ca2+affects the nacre deposition of H. cumingii, and play the key roles in regulation of nacreous formation.