Isolation,Identification And Functional Analysis Of Shell Matrix Protein Gene Hic22 And Hic74 In Hyriopsis Cumingii

Hyriopsis cumingii, Chinese unique freshwater mussel, accounts for 80 percent of freshwater pearls. Previous studies have shown that matrix proteins played roles throughout the whole process of shell and pearl formation. Studies about matrix proteins mainly focused on seawater shellfish, while matrix proteins from freshwater shellfish shell were less reported. In this study, two matrix protein genes hic22 and hic74 related to nacreous layer formation were cloned and characterized.A 22 kDa matrix protein was identified and characterized from the nacreous layer of H.cumingii, and its N-terminal amino acid sequence was determined. Degenerate oligonucleotide primers were designed based on its N-terminal amino acid sequence, and another group of primers based on the amino acid sequence “AAAAAAA” of MSI60. With the two group primers, two novel matrix protein genes hic22 and hic74 were cloned and characterized from the mantle of freshwater mussel H.cumingii. Firstly, the full length cDNA of hic22 is 731 bp with a 621 bp open reading frame. Hic22 encodes a 22 kDa protein composed of 185 amino acids. The full cDNA sequence of hic74 is 2882 bp with a 2553 bp open reading frame. Hic74 encodes a 73.7kDa protein composed of 850 amino acids. The deduced amino acid sequence revealed a modular structure including a high proportion of Ala(30.8%), Gly(25.6%) and Ser(10.6%) with Ala mainly existing in poly-Ala forms in the Ala/Gly-rich regions and there are three acidic regions in hic74. Tissue expression analysis indicated that hic22 and hic74 are specifically expression at the mantle, and in situ hybridization indicated that hic22 and hic74 are expressed in the dorsal epithelial cells of the mantle pallial. All of the results imply that matrix proteins hic22 and hic74 might account for nacreous layer formation.In pearl-nucleus-inserting experiment, the results observed by the scanning electron microscope(SEM) showed that the process of CaCO3 deposition during pearl formation could be divided into two consecutive stages: the CaCO3 primitive accumulation and the CaCO3 regular deposition. Before the nacreous layer formation, the CaCO3 coats the nucleus irregularly to make it have a smooth surface. During the nacreous layer formation, CaCO3 nucleates in the surface and deposited regularly. The quantitative real-time PCR(qRT-PCR) revealed that the expression levels of hic22 and hic74 were low throughout the period of CaCO3 primitive accumulation. The expression level increased significantly at nucleation period, and rose gradually during the crystal growth period, revealing that matrix proteins hic22 and hic74 took part in the CaCO3 nucleation and regulated the growth of aragonite crystals during pearl formation.In the shell repair experiment, the SEM results showed the periostracum reform at the breakage, and CaCO3 crystals were deposited on the periostracum to form the prismatic layer. Once the prismatic layer has formed, the aragonite crystals quickly covered the prismatic layer, and the crystal tables have a staggered “brick wall” structure. Compared with the control groups, the expression of hic22 in experimental group was lower with a sustained downward trend, revealing that hic22 was jointly involved in shell biomineralization with other matrix proteins. Then, high expression level of hic74 in the rapid growth of prismatic and nacreous layers imply hic74 may account for prismatic and nacreous layers formation.