| Pinctada fucata is one of the species to study the mineralization mechanism of shellfish since its nacreous layer has excellent performance.The principle of shell formation is one of the research hotspots in this field.In the previous study,the transcription level of each gene in different developmental stages was determined by gene chip,and several matrix protein candidate genes were screened according to the characteristics of matrix proteins,however,their functions need further identification.In this research,one of these unigenes was functionally identified and we firstly analyzed the effects of mantle protein N25(N25)on mineralization.The full length of N25 was obtained by RACE,which was specifically expressed in the mantle tissue.After purifying the inclusion bodies of N25,the active soluble protein was obtained by protein refolding.A scheme for obtaining poorly soluble matrix proteins by renaturation of inclusion bodies was preliminarily established.The purified N25 has the ability to bind calcite,aragonite,and chitin.Immunoblotting of the shell fraction revealed N25 was distributed in the insoluble component of both the nacre and prism layer.N25 could alter the morphology of both calcite and aragonite.We used a SIM microscope to image the distribution of cy5-labeled N25 on calcite.The results show that N25 protein is only distributed on the crystal surface.With the help of the simulation method,we obtained the predicted calcite morphology.We speculated that N25 protein regulates the calcite shape by binding to the specific crystal planes and reducing the attachment energy.We traced the secretion of N25 by eukaryotic expression of N25-EGFP protein and observed the secretion by the vesicle.By using SDS and DTT to extract the insoluble framework of the nacre and the prism layer,we eluted different matrix protein components.The N19 protein is rich in Cys and is dissociated from the framework during DTT treatment.It is speculated that N19 may be linked to other macromolecules via disulfide bonds.Some framework proteins produce a non-covalent linkage to the framework with a chitin-binding domain that is easier to dissociate after SDS treatment.Some matrix proteins have neither the Cys nor the chitin-binding domain but they could still bind to the framework firmly.These phenomena indicate that the composition and assembly of the shell frame are multi-layered and complex. |
