Identification Of Two Matrix Protein Genes Hic31 And Hic52 From Hyriopsis Cumingii And Their Functional Exploration On Biomineralization

Hyriopsis cumingii, a unique freshwater mussel of China, dominates a very important role in pearl indurstry for producing 80% of freshwater pearls in the world. And it is well known that the shells and pearls of H.cumingii are the products from biological mineralization, the process of depositing calcium carbonate crystals under the control of various matrix proteins. The identification and functional exploration of matrix proteins can help to understand the biomineralization mechanism of shells and pearls. Most researches about the matrix proteins are focused on seawater mussels, while the reports from freshwater mussels are less known. In this study, two matrix protein genes, hic31 and hic52, were isolated and identified from H. cumingii, then structural and functional characters were analyzed. 1. The cloning of hic31 gene from H. cumingii and its expression pattern analysisThe experimental analysis, including sequence analysis, structural prediction, differential expression profile of tissues and in situ hybridization were applied to hic31 gene of H. cumingii. The full-length cDNA sequence of hic31 gene obtained by RACE is 1432 bp with an open reading frame of 957 bp encoding 318 amino acids. Regardless of the signal peptide of 1-18 amino acids, the calculated molecular weight is 28.82 kDa and the PI is 7.0. Sequence composition analysis of amino acids revealed that it had a high proportion of glycine residues(26.67%), and glycine residues are frequently clustered as multiple polyglycine blocks((Gly)n(n>2)) in the N-terminal region. Meanwhile, the longer poly glycine blocks in other regions are frequently subdivided by Met or Ser, leading to multiple repeats of(Gly)m X(Gly)n(m>1, n>1, where X prefers to Met or Ser). Plus, secondary structure prediction indicates that hic31 is mainly composed of α-helix, and its tertiary structure is similar to that of collagen type I, α1 and α2. Meanwhile, the expression pattern analysis shows gene hic31 is specially expressed in mantle tissue, and its expression occurs primarily at the edge rather than the pallial region. In situ hybridization revealed strong signals in the epithelial cells at the mantle edge. All these features mentioned above implies that hic31 are supposed to be associated with prismatic-layer biomineralization as a framework-matrix protein. 2. The cloning of hic52 gene from H cumingii and its expression pattern analysishic52 gene was identified and analysed. The full-length cDNA sequence of hic52 from RACE was 2053 bp. The open reading frame(ORF), with the length of 1629 bp, encodes 543 amino acids in total. Apart from the signal peptide of 18 amino acids, the calculated molecular mass of the polypeptides is 52.2kDa and the predicted isoelectric point is 10.37. Sequence analysis reveals that hic52 comprises a high proportion of Gly(28.8%) and Gln(12.4%). Secondary structure prediction by Phyre2 indicated that hic52 protein carried with a long α-helices in the end of C-terminal, and the tertiary structure shared some similarities with that of collagen, type I, alpha 1 and alpha 2 as well. In addition, hic52 gene is specially expressed in mantle tissue especially at the mantle center, which was in accordance with the result of in situ hybridization on frozen mantle sections strong signals occuring at the dorsal epithelial cells of mantle center. Thus, it can be concluded that hic52 was probably a framework shell protein, involved in the nacreous layer biomineralization process. 3. The functional analysis of hic31 and hic52 genes in pearl formation processImplantation experiment of pear sacs and optical microscope observation of pearl particles from H.cumingii were conducted. To further investigate the function of hic31 and hic52 genes in pearl biomineralization, pearl sac tissues and small pearl particles from healthy individuals after implantation were sampled regularly, of which the pearl sacs was prepared for qRT-PCR analysis and small pearl particles for microscope observation. As a result, qRT-PCR indicated that, apparently, the expression of hic31 gene during early pearl sac development increased during early stages, and decreased after day 23 until no expression was detected on day 77, and that the expression of hic52 remains low before 12 d, during day 12-23, the expression relatively increased, whereas after day 23, the expression keeps increasing until a low expression level was observed at day 77. The obvious lustre was observed firstly in particles on day 23, comprehensively indicating that protein hic31 is mainly associated in the prismatic layer biomineralization and that protein hic52 primarily play aimportant role in the nacreous layer biomineralization.