| H.cumingii is an unique freshwater mussel in China.Its pearl production has reached over 80% of the world’s total pearl production.Studies have shown that the matrix proteins play an important role in the formation of shells and pearls as an important component in the organic composition of shells and pearls.The identification and analysis of matrix proteins were of great significance to the efficient development of pearl culture industry.In this study,two shell matrix protein genes were isolated and identified from H.cumingii.Their structure and function were analyzed.Two matrix protein genes hic24 and Hc-temptin were cloned from the mantle of H.cumingii.The total length of cDNA sequence of hic24 gene is 756 bp,with an open reading frame of 654 bp,theoretical molecular weight of 23.5 kDa and an isoelectric point of 6.36,which is an acidic matrix protein.It encodes 217 amino acids and includes a signal peptide of 18 amino acids.The prediction of secondary structure showed that it was mainly a β-sheet structure,and the amino acid composition of the sequence was rich in Gly(11.5%).The full length of cDNA sequence of Hc-temptin fene is 884 bp,containing a 411 bp open reading frame(ORF),encoding 136 amino acids.The theoretical molecular weight was 14.7 kDa and the isoelectric point was 7.60.The first 21 amino acids were predicted to be signal peptides,and the amino acid composition was rich in Ser(10.3%),Asn(10.3%),and Gly(9.6%).Through tissue fluorescence quantitative analysis,two matrix protein genes hic24 and Hc-temptin were specifically expressed in the mantle membrane tissue.In situ hybridization experiments,hic24 gene was specifically expressed in the epithelial cells of the central membrane of the mantle,and Hc-temptin gene was specifically expressed in the dorsal epithelial cells of the mantle pallia.It is speculated that the hic24 gene is involved in the formation of the shell nacreous layer,and the Hc-temptin gene plays an important role in the formation of the shell prismatic layer.In the RNA interference experiment,the function of the two matrix protein genes was quantitatively detected in the mantle membrane after interference by fluorescence injection of 20 μg/100 μL and 40 μg/100 μL of dsRNA.The results showed that the expression levels of hic24 and Hc-temptin genes were significantly decreased in the mantle.Under the observation of scanning electron microscope,the hic24 gene had an effect on the structure of the nacreous layer,while the structure of the prismatic layer did not change significantly.It is speculated that the hic24 gene was involved in the regulation of nacre nucleation and crystal morphology and growth.The Hc-temptin gene has an impact on both the prismatic layer and the nacreous layer structure.It is speculated that the Hc-temptin gene was involved in the regulation of the morphology and growth of the prismatic layer and nacreous layer.The effects of two matrix protein genes in the formation of pearls were explored by insert experiments.The pearl formation process involves two successive stages: disordered deposition and ordered deposition.The quantitative detection of two genes in the formation of pearl sac showed that the expression levels of the genes hic24 and Hc-temptin were lower in the disordered deposition stage,but the expression level was significantly increased in the ordered deposition stage.It is speculated that the hic24 and Hc-temptin genes regulate the growth of nacreous layer.In pearl-nucleus-inserting experiment,the newly grown shells and mantle were observed and analyzed by SEM electron microscopy and fluorescence quantification respectively to study the role of hic24 and Hc-temptin in shell mineralization.During the repair process,it can be observed that the stratum corneum is formed at the crack,and then the prismatic layer begins to accumulate and finally form a nacreous layer.In the fluorescence quantitative results,the expression level of hic24 gradually increased during the growth of the prismatic layer and the nacreous layer,and was higher than that of the initial expression.The gene hic24 may be played a role in the growth of the prismatic layer and the nacreous layer.Compared with the expression level at the expression level of Hc-temptin gradually increased in the growth phase of the shell prismatic layer.The expression level tended to be the expression level of the control group in the growth of the nacreous layer.The gene Hc-temptin may be involved in the growth of the shell prismatic layer and interacts with other matrix proteins on the growth of the nacreous layer. |
