The Shell Proteomics And Stress Network Of Biomineralization In Pearl Oyster

Biomineralization refers to an orderly deposition of mineral crystals(biological minerals) internal of the organism under the control of bio-organic molecules(primarily proteins, glycoproteins or polysaccharides). Molluscan shell is a typical representative of biological minerals produced by mollusk species. Pinctada fucata martensii is a major sea pearl shell in China as well as in Japan. To uphold the quality and market value of the pearl, we detected and analyzed of matrix protein(or protein family) contained in the prismatic and nacreous layer of shell based on fine pearl oyster genome. Meanwhile, we screen significantly response gene module(s) and construct a biomineralization-related gene network under non-physiological conditions by using WGCNA. Specific contents are as follows: 1. Shell proteomics in Pinctada fucata martensiiBy using LC-ESI-MS/MS, we identified acid insoluble( PI) and soluble proteins(PS) in prismatic layer, nacre insoluble(NI) and soluble protein(NS) respectively, as 207,109,199 and 120 in number. Of the total 366 proteins, 78 are known functional proteins. The proteins content of top 20 in the 4 samples were calculated. Tyrosinase(Tyr), fibronectin like protein(FLP) and nacre protein N19 are found in all samples. Related low complexity domain(RLCD) proteins, also known as intrinsically disordered proteins(IDPs), are high level in nacre and prism layer. The nacre matrix proteins such as the N19, nacrein were identified in all four samples, but the levels were difference. The same situation also occurs in the prismatic matrix protein, such as Tyr, FLP. In combined with the gene expression data, the conclusion of multi-tissue contribution to crystal specific gene expression is in contradict with the traditional understanding of the mantle regions for different partitions. Finally, we compared shell matrix proteins of three molluscan species. Novel function gene nacrein derived from ancient carbonic anhydrase was detected in all species. Multi-copy genes include Tyr family and ECM coding genes which indicate their important roles in shell biomineralization.2. Shell protein(family) sequence analysis, homology modeling and functional prediction in Pinctada fucata martensiiClone whole length cDNA 10 Crassostrea gigas Tyrs and 2 P. fucata martensii Tyrs. This study focuses on those Tyr family with high abundance in shell which indicate p I may be related to its biomineralization function. Phylogenetic analysis indicates function differentiation may have occurred. Homology modeling structure of Tyr located the key amino acid in the catalytic center, and their relation with the Tyr functions and. Another high level matrix protein family are FLPs with 4 tandem FNIII domains. These FNIII domains contain different amino acid repeat but were lack of important functional domains RGD found in mammal. Phylogenetic analysis indicate the FNIII in FLPs were homologous to those in protein tyrosine phosphatase(PTP) of high animals. Sequence and structure analysis FNIII family show the, revealing its function conversion. IDPs are abundance in four samples. On the genome level, IDPs proportions of total protein shell in three molluscan species were 28.53% in P. fucata martensii, 15.55% L. gigantea and 26.64% in C.gigas. Species matrix proteins are relative higher proportion of IDPs with 34.23%、18.52%�'� 34.62% respectively. Two types of IDPs were classified by length of the conserved region. One is including those that are highly homologous in full length of sequences, such as nacre matrix protein MRMP34, NUSP1 /2/5/7/8/9 and the prismatic matrix protein PUSP2 /8/10. The other are those contain conserved motifs such as pearlin(N16), nacrien, MSI60, which contain crystal nuclear site motif DDNGN of abalone nacre crystalline matrix protein AP7. Conservative motifs in nonhomologous aragonite matrix proteins represent a common type of modular function. 3. The network construction for high temperature and ocean acidification stress in pearl oyster by co-expression network analysis(WGCNA) Gene expression data of pearl oyster under high temperatures and seawater acidification stress from NCBI website CEO database were analyzed by WGCNA. One gene module of heat stress and five modules were screened. GO enrichment of those modules indicates those functional genes cluster response to environmental factor. Cytoskeletal proteins such as ARHGEF4, SAP and myosin were associated to temperature stress. Acidification transponder modules recruit heme A synthase, aromatic amino acid metabolism-related enzyme, cadherin and platelet-reactive protein. We also focus on signaling pathways in those modules, including the heat shock pathway. Among these signaling pathways, membrane receptors are sensitive. Interestingly, the high stress module enriched the most mineralization genes, including Tyrs and FLPs. To reveal the relation between the mineralization genes and other function genes, we constructed the gene co-expression network, showed that calcium-binding proteins, cytoskeletal proteins and protein glycosylation genes closely related. Simulated the condition of ocean acidification,we observed changes in nacre crystal morphology and examined the expression levels of nacre mineralization associated genes(pif177, nacrein and pearlin) in the mantle pallial zone.