Preliminary Study On The Role Of Shell Matrix Proteins In Nacre Biomineralization And Their Influence Factors In Hyriopsis Cumingii

After the successful development of artificial non-nuclear pearl cultivation in the late 1960s,freshwater pearl culture industry has been maturation through more than fifty years development in China.Because of the degeneration of genetic characterization,the deterioration of ecological environment and short pearl-cultivating time,the output value of non-nuclear pearls is very low.Mussels absorb inorganic ions from the external water environment and soft tissues of the food to form a highly structurally ordered and mechanically exceptional shell and pearl under the control of organic matrix secreted by mantle and pearl sac.The researches on the functions of matrix proteins and their influence factors have significant meanings for a better understanding of formation mechanism of pearls and further improving pearl quality.In this paper,Chineses important freshwater pearl mussels Hyriopsis cumingii were selected as our experimental animals.The roles of shell matrix proteins in nare biomineralization were explored on molecule and protein levels.The correlations were detected between shell matrix protein gene expressions and pearl weight.The effects were evaluated about temperature variation and pearl sac cells transdifferentiation on nacre deposition and shell matrix protein gene expressions.Main results were as follows:1.Cloning and functional analysis of shell nacreous layer matrix protein genes from H.cumingii.A novel matrix protein gene?teosin?was isolated from the mantle of H.cumingii.The full-length cDNA of teosin was 522 bp encoding a 70-amino acid protein,and1-19 amino acids in N-terminal was signal peptide sequence.N-terminal of mature protein was hydrophobic domain and C-terminal was hydrophilic domain.Teosin was characterized by a high proportion of Gly,Cys,and Pro residues with Cys mainly located in hydrophilic domain and a predominant?-sheet conformation according to secondary structure prediction.Gene-expression analysis showed that teosin was mainly expressed in the mantle and blood.The hybridization signal were detected in dorsal epithelial cells of the mantle pallial.In RNAi assay,the suppressing teosin expression resulted in disordered arrangement of irregular crystal and growth inhibition of nucleated seeds.In vitro crystallization assays,teosin could increase the size of calcite,and induce polycrystal formation.These results suggested that teosin was involved in regulating crystal morphology regulation and inducing polycrystal formation during shell nacreous-layer formation.A matrix protein gene HcKuPI was cloned and characterized from H.cumingii.The full length of HcKuPI cDNA was 543bp with a 333bp open reading frame encoding 110 amino acids.Regardless of the signal peptide of 1-24 amino acids,the deduced amino acid sequence included a high proportion of Lys?20.9%?,Gly?12.8%?and Ser?10.5%?,and the calculated molecular weight was 9.5kDa and theoretical isoelectric point?pI?was 9.95.The deduced amino acid sequence of HcKuPI revealed a modular structure including N-terminal signal peptide,Lys-rich region and C-terminal Kunitz domain.Tissue expression analysis and in situ hybridization results indicated that HcKuPI was specifically expressed in the dorsal epithelial cells of the mantle pallial.HcKuPI dsRNA injection caused an irregular surface and disordered deposition on the aragonite tablets of the nacreous layer.These results indicated that HcKuPI played a vital role in shell nacreous layer biomineralization.The recombinant protein GST-HcKuPI was expressed mainly in supernatant,and in vitro calcium carbonate crystallization assay,the addition of GST-HcKuPI increased the precipitation rate of calcium carbonate and induced the crystal overgrowth of calcium carbonate.These results indicated that HcKuPI participated in calcium carbonate deposition acceleration and morphological regulation of the crystals during nacreous layer formation.2.Expressions of shell matrix protein genes in the pearl sac and its correlation with pearl weight in the first 6 months of pearl formation in H.cumingiiCaCO₃ deposition was analyzed during the first month of pearl formation and the expression patterns of twelve shell matrix protein genes?Hcperlucin,hic31,silkmapin,hic22,hic24,hic52,hic74,HcTyr,HcCA3,Hc-upsalin,HcKuPI and teosin?were examined in the pearl sacs of H.cumingii.During pearl formation,CaCO₃ crystals were initially deposited in a disorderly manner on day 15.On day 18 and 21,CaCO₃crystals gradually nucleated on an organic membrane,and the pattern of crystal deposition changed significantly.Between days 24-30,crystals similar to nacre tablets were deposited,then grew and formed connections in a more orderly fashion,eventually forming the nacreous layer.The high expression of prismatic layer related gene hic31 occurred during disordered crystals deposition.The high expressions of silkmapin and teosin occurred during transition phase.The high expressions of nacreous layer related genes occurred during nacre tablets deposition.These results indicated the crystal deposition during pearl formation was under the control of shell matrix proteins.The correlations were analyzed between the expression patterns of matrix protein genes and pearl weight during the first six months after grafting.The prismatic-layer matrix protein hic31 and nacreous-layer matrix protein hic22 shared negative correlations with pearl weight.Silkmapin and teosin had no significant correlations with pearl weight and other eight nacreous-layer matrix proteins had significant positive correlations with pearl weight.These results indicated shell matrix proteins controlled the crystal deposition during pearl formation and further determined pearl quality traits.3.Effect of temperature on pearl nacre deposition rate and matrix protein gene expressions in H.cumingiiThe recipient mussels were cultivated in a temperature gradient?16?,20?,24?,28?and 32??for a month.There were no visible pearl granules collected at16?and 20?groups.At 24?group,several small tawny pearl granules were collected and SEM result showed an organic membrane covered upon the pearl surface.At 28?and 32?groups,lustrous pearls with nacre aragonite tablets deposition were collected.The relative expression patterns of hic31 and silkmapin increased significantly and other nacreous layer matrix proteins kept low expression at day 28 at 16,20 and 24?groups.At 28?group,expressions of hic31 and silkmapin peaked and other nacreous layer matrix protein genes?hic22,hic24,HcTyr,Hc-upsalin?significantly increased at day 28.At 32?group,expressions of hic31and silkmapin peaked on day 14.Other nacreous layer matrix protein genes?hic22,hic24,HcTyr and Hc-upsalin?significantly increased at day 28.These results indicatd that raising the temperature could promote pearl sac and pearl formation.Seasonal variations of water temperature also had a significant influence on nacre deposition rate?NDR?and expressions of matrix protein genes.Among the four seasons,the NDR of pearl was the lower in winter and faster in summer.The NDR of pearl was higher in June and August,the corresponding temperature were 28.3?and 30.3?respectively.qRT-PCR results showed expression levels of hic31,silkampin,teosin and hic22 were higher in winter than other seasons.Other nacreous layer matrix proteins reached significantly higher expression in higher water temperature seasons.These results indicated water temperature affected NDR,and NDR was highest within the temperature range 28.3?-30.3?.Water temperature also affected the expressions of shell matrix protein genes.4.Effect of cell transdifferentiation of pearl sac on pearl nacre deposition and matrix protein gene expressions in H.cumingiiIn grafting operation,the epithelial cells of mantle pallium were divided into edge and pallial epithelial parts,and made into saibos to transplante into recipient mussels.SEM showed 3-month-old pearls secreted by pallial epithelial cells pearl sac?PECPS?were lustrous with regular nacre crystal deposited on the organic membrane.The pearls secreted by edge epithelial cells pearl sac?EECPS?were brown and lusterless with needle-like crystals deposited on organic membrane.The pearls of6-month-old secreted by PECPS were covered by regular aragonite tablets.The pearls secreted by EECPS showed calcium carbonate crystals nucleated on the dimples of prism columns and regular aragonite tablets gradually covered the prisms.The nacreous tablets were filled within the prisms in cross section observation.The expressions of matrix protein genes in EECPS showed nacreous-layer matrix protein genes expressed significant higher on 6 month than 3 month.These results indicated cell transdifferentiation may have taken place in EECPS in 6 month,and EECPS began to secret nacreous layer matrix proteins to control nacre tablets deposition.