Studies On Biomineralization Of Shell Matrix Protein-silkmapin Of Hyriopsis Cumingii

Hyriopsis cumingii, a most important pearl mussel of China, produced 95% of freshwater pearls in the world. Understanding the role of matrix proteins of mollusks could provide meaningful information for the mechanism of nacre formation and biomineralization of shell or pearl. The known shell matrix proteins were mostly isolated and identified from seawater mussel, while matrix proteins from freshwater mussel shell were less discovered and reported. In this study, the gene encoding the novel H. cumingii shell matrix protein silkmapin was characterized, and its structure and function were studied.1. The cloning, sequence analysis, structure prediction, differential expression analysis of tissues and in situ hybridization were conducted about silkmapin of H. cumingii. The full c DNA sequence of silkmapin was 1242 bp with an open reading frame of 978 bp determined by RACE technology. Sequence analysis revealed that the gene encoded a protein of 30.89 k Da, and that Gly accounted for 34.41% of the total amino acid content and most Asp residues of the protein were concentrated on the C-terminal region which could bind Ca2+. By secondary structure prediction, β-fold was predominantly found and the protein owned a typical structure of filamentous proteins. Real-time quantitative PCR showed that silkmapin was mainly expressed in epithelial cells at the edge and pallial of mantle tissue. With In situ hybridization, the result was same as obtained from real-time quantitative PCR. A strong signal was detected in the epithelial cells at the mantle edge and pallial region. These results of expression analysis indicated that silkmapin probably played a role in the nacreous and prismatic layer formation of shell.2. The role of silkmapin was studied in the shell biomineralization with shell repair experiment. The live H. cumingii with artificially damaged shell was set as treatment group, and two control groups were designed. The shells of new growth and mantle tissues corresponding to the damaged part were sampled for further anlysis. The new shells were inspected by SEM to observe the dynamic process of shell growth, and mantle tissue samples were applied for gene expression analysis with q RT-PCR method. SEM showed that the shell of H. cumingii grew from the outside to inside layer by layer. Besides, the growth followed a order of periostracum-prismatic layer- nacreous layer. At first, a thin periostracum was formed, then the aragonite crystal grain of prismatic layer grew on the thin periostracum, which could develop both horizontal and vertical directions. Once the prismatic layer matured, the sheet hexagonal aragonite nacre crystal began to pile up on it. No obvious transition period was observed during the growth of the prismatic layer to nacreous layer. The growth of prismatic layer conformed to the feature of "squeeze hypothesis", and the nacreous layer is a typical "brick-type" structure. Combined with the results of SEM and q RT-PCR, it was found that the gene expression of experimental group during the growth of prismatic layer and nacreous layer was much higher than the two control groups of the same stage. It was indicated that silkmapin indeed played important roles in biomineralization of prismatic and nacreous layer formation of H. cumingii.3. The function of silkmapin in the pearl biomineralization was further investigated by grafting the saibos into mantles of healthy H. cumingii of equal size. Pearl sac tissues and small pearl particles were sampled regularly. The pearl sac tissues was prepared for q RT-PCR, and small pearl particles for SEM. SEM exhibited that the pearl biomineralization process in H. cumingii, could fall into two stages, calcium carbonate disorder deposition and ordered deposition. During these two periods, evident changes of surface morphology could be viewed, which became from the uneven, messy piled morphology at the begaining to later smoothy, orderly stacked status. With the results of SEM and q RT-PCR, it was found that the gene expression of silkmapin showed a gradually upward trend in pearl nucleation stage. The expression of silkmapin in the period of calcium carbonate ordered deposition was much higher than that of calcium carbonate disordered deposition, and the highest expression level was appeared in the initial formation period of nacreous layer. It was illustrated that silkmapin was not only involved in the formation of nacre essential calcium carbonate nucleation process, but also regulated the stack of small nacre aragonite pieces.