| The triangle sail mussel(Hypriosis cumingii)is a freshwater pearl breeding mussel in China that is used to cultivate freshwater pearls,and its freshwater pearl production accounts for about 90% of the country’s freshwater pearl production.The shell is the result of the biomineralization process,which functions as an exoskeleton providing effective protection to various tissues inside the organism.As organic matrices direct and regulate the deposition of calcium carbonate,shells and pearls have incomparable physicochemical and biological properties.Therefore,the interrelationship between organic matrices and mineral crystals has become a focus of research in materials,medicine and biology.The identification and investigation of the mechanism of matrix protein roles in mussel shell and pearl formation will help us to understand their formation mechanisms in depth.In this study,three novel matrix protein genes,low complex domain protein(LCDP),Hc-fibrillin,and Hc-transgelin,were identified in the mantle of H.cumingii,and their mechanisms of action in the biogenesis of H.cumingii were investigated.The specific research results are as follows:1.Cloning and expression analysis of LCDP,Hc-fibrillin and Hc-transgelin genes in shell of the the triangle sail mussel H.cumingii.The cDNA of LCDP was 1058 bp,ORF was 714 bp,encoding 237 amino acids,Gen Bank accession number: OP747293.LCDP was rich in Gly(24.10%)and the secondary structure predicted that LCDP contains 50% β-fold and 5% α-helical structure containing a low-complexity structural domain.q RT-PCR and semi-quantitative PCR showed that LCDP was specifically expressed on the mantle.Based on the in situ hybridization results,LCDP was found to have a significant positive signal at the rim membrane of the mantle.The cDNA of Hc-fibrillin was 1952 bp,ORF was 1389 bp,encoding 462 amino acids,Gen Bank accession number: OM223075.The secondary structure of Hc-fibrillin gene was mainly β-folded and contained 8 calcium-binding epidermal growth factors(EGF-Ca structural domain).the expression of Hc-fibrillin was significantly higher in the mantle and gill than in other tissues.In situ hybridization showed that Hc-fibrillin was expressed in both epithelial cells of mantle.The cDNA of Hc-transgelin was 1200 bp,ORF was 501 bp,encoding 166 amino acids,Gen Bank accession number: OM937127.Hc-transgelin was significantly expressed in the mantle and foot.2.The role of LCDP,Hc-fibrillin and Hc-transgelin,the shell matrix protein genes in shell mineralization of the the triangle sail mussel H.cumingii.Mollusks have a self-repair mechanism to achieve shell regeneration after shell damage,and the whole repair process is similar to shell formation.In the shell-breaking repair experiment,q RT-PCR results showed that the expression of LCDP and Hctransgelin increased significantly in the experimental group from day 12,while the expression of Hc-fibrillin increased significantly from day 16,and as the experiment continued,the expression of LCDP in the experimental group was no longer significantly different from that of the control group at day 25,while the expression of on day 28,the expression of Hc-fibrillin and Hc-transgelin returned to the level of the control group.After silencing the three genes separately,the expression of all genes decreased by more than 70%.The normally grown pearl layer was dominated by small hexagonal aragonite fragments with smooth surface and nucleation sites,and the prismatic layer had a smooth surface with crystals closely connected to each other by organic matrix.In the ds RNA-Hc-fibrillin group,the aragonite fragments in the pearl layer tended to be rounded,and the surface of calcium carbonate crystals in the prismatic layer was extremely rough,with great differences in size,and the organic matrix between the crystals appeared skeletonized;in the ds RNA-LCDP group,the aragonite fragments were disordered and stacked,with no obvious features in morphology and size,while the surface of calcium carbonate crystals in the prismatic layer was rough;in the ds RNA-Hc-transgelin group,there were obvious calcium carbonate deposits on the edges of aragonite fragments and the organic sheaths between the prismatic layer crystals showed obvious cracks.3.Investigation of the effect of matrix protein Hc-fibrillin and LCDP on calcium carbonate crystals in the shell of the the triangle sail mussel H.cumingii.Generally,the pearl layer of mussel shells is composed of small pieces of aragonite and the prismatic layer is calcite.It is worthwhile to investigate why calcium carbonate is deposited so precisely in the same body.this chapter verifies the effect of Hc-fibrillin and LCDP peptides on calcium carbonate crystallization by reacting synthetic peptides with saturated calcium bicarbonate solution.In the experimental group of Hc-fibrillin peptide,calcium carbonate crystals were transformed into isolated needles,and according to the results of Raman spectroscopy,calcium carbonate crystals were found to be transformed from calcite to spherical aragonite.In the LCDP peptide experimental group,the calcium carbonate crystals showed a polycrystalline form,and the Raman spectroscopy results showed that no change was found in the calcium carbonate crystals,which were merely single crystals stacked into polycrystals. |
