Identification And Functional Studies Of Shell Matrix Protein Gene Cysrichin、GYSRIN From The Hyriopsis Cumingii

Through tens of billions of years of natural selection and evolution,the structure and function of organisms have been constantly improved;its structure and function reached a high degree of coordination and unity.The precise and regular microstructure of shell is a typical representative of biomineralization,and the organic matrix in the shell is considered as the core role of biomineralization.Scientists have done a lot of research on organic matrix,especially matrix protein components.Recently,a large number of matrix proteins and their functions have been identified in mollusks,and they participate in calcium carbonate precipitated,crystal arrangement and crystal morphology during shell formation of mollusks.However,the complex mineralization process has not been completely revealed.In this study,two matrix protein genes,named cysrichin and GYSRIN,were identified from the mantle of Hypriosis cumingii,the experiments were performed to research the function of cysrichin and GYSRIN in mussels biomineralization.The results are as follows:1.Identification and functional study of matrix protein cysrichinThe cysrichin gene was cloned from mantle of hyriopsis cumingii by using degenerated primers which designed based on the amino acid sequence "DAGFS" of Nacrein from Pinctada fucata.The cysrichin cDNA full-length was 648-bp,containing an open reading frame of 492-bp,encoding 163 amino acids.The theoretical molecular weight of cysrichin protein was 17.53 KDa and the theoretical isoelectric point was 8.15.The cysrichin protein is richin cysteine(14.7%),serine(11.7%)and glycine(8.6%)residues,and is similar to the coral biomineralized protein galaxin in protein structure.Tissue expression analysis showed that cysrichin was mainly expressed in gill and mantle.Further in situ hybridization showed that cysrichin m RNA was strongly expressed in the whole epithelial of mantle tissue.RNA interference analysis showed that when cysrichin expression was inhibited,the morphology of calcium carbonate in nacreous layer was significantly degraded,and the normal formation of organic framework in prismatic layer was affected.Shell notching results showed that cysrichin maintained a high expression level during shell regeneration.Above results incicated that the matrix protein cysrichin plays an important role in the shell biomineralization and participates in the regular growth of the nacreous and prismatic layers of hyriopsis cumingii.2.Identification and functional study of matrix protein GYSRINIn this study,a novel matrix protein GYSRIN was cloned from mantle of hyriopsis cumingii by degenerating primers based on amino acid "GYSGYS".The full length of GYSRIN gene was 418-bp with an open reading frame of 207-bp,encoding68 amino acids and containing a 17-amino acid signal peptide.Without the signal peptide,GYSRIN protein was rich in glycine(31.4%),tyrosine(21.6%)and serine(17.6%)residues,with a theoretical molecular weight of 5.53 KDa and a theoretical isoelectric point of 10.22.Tissue expression analysis showed that GYSRIN was strongly expressed in the mantle,and in situ hybridization showed obvious hybridization signals in the center region of mantle epithelium.When GYSRIN expression was inhibited by 60%,the growth of aragonite crystals in the pearl layer of shell was inhibited,and the aragonite crystals were arranged irregularly.There is no obvious change in prismatic layer morphology.During the process of shell regeneration,the expression of GYSRIN increased gradually and maintained a high level.The amino acid composition and structure of GYSRIN are similar to silklike protein,and GYSRIN may be involved in the organic skeleton during shell formation and control the precise growth of aragonite.3.Effects of matrix proteins cysrichin and GYSRIN on crystal form of calcium carbonate in Hyriopsis cumingiiBy mixing protein solution with saturated calcium bicarbonate solution,the effect of matrix protein on the crystal shape of calcium carbonate can be investigated.In this study,the synthesized cysrichin and GYSRIN polypeptides were mixed with saturated calcium bicarbonate solution as experiment group,while the control group was pure saturated calcium bicarbonate solution.Raman spectroscopy and scanning electron microscopy showed that calcite crystals were formed in the control group,while "lotus" shaped calcium carbonate crystals were formed under the induction of cysrichin polypeptide,and flat spherical calcium carbonate crystals were formed by the addition of GYSRIN polypeptide solution.These results indicate that matrix proteins cysrichin and GYSRIN can induce the change of crystal morphology of calcium carbonate,and play an important role in shell mineralization.