Proteomic Analysis Of The Shell Matrix Of Perna Viridis And Functional Analysis Of Transgelin And Whirlin Proteins Of Mytilus Coruscus

Biomineralization is a process in which organisms deposit minerals and form hard tissues,such as skeletons and shells.Among them,the shells of mollusks and snails are typical biomineralization products.Its main component is composed of CaCO₃ crystal,matrix protein and other biomolecular complexes.Compared with pure mineral structure,the shell shows more excellent hardness and strength.Although the content of organic matter in shell is less than 5%,the organic matrix,especially the protein of shell matrix,is considered to be directly involved in the nucleation,polymorphism,orientation and morphological growth of calcium carbonate crystal.At present,more and more shell matrix proteins（SMP）of shellfish have been described at the level of histology.At the same time,it is found that there are differences in the composition of SMP among different microstructure levels in the shell,which indicates that different structural levels in the shell may be formed under the guidance of different SMP.The shell of Perna viridis is mainly composed of aragonite type calcium carbonate crystal.The shell microstructure mainly includes nacre layer and myoprism layer.Compared with the shell microstructure of other shellfish,the shell of Perna viridis has the characteristics of simple structure and single crystal type of calcium carbonate,so it is an excellent material to study the shell formation mechanism.In order to understand the shell microstructure and SMP composition of Perna viridis,the different shell structures were observed by FTIR,SEM and XRD.Through the method of combining transcriptome and proteomics,the proteomic analysis of the shell matrix protein of Perna viridis was carried out,in order to find out the characteristics of protein molecular composition in different microstructure levels of Perna viridis.The myoprismatic layer of mussel is the main part of the connection between muscle and shell,but it is not clear how SMP from myoprismatic layer can play a role in biomineralization and muscle shell interface.In order to explore the regulatory role of SMP in the formation and development of Mytilus,we studied the specific whirlin like protein（WLP）with PDZ domain and Transgelin like protein 1 with CH domain In order to study the role of WLP and TLP-1 in biomineralization of shell,especially in myoprismatic layer,the recombinant expression,functional exploration and location analysis of WLP and TLP-1 protein were carried out.The results show that the shells of Perna viridis have two different mineral layers,myoprismatic layer and nacreous layer,and have the same aragonite calcium carbonate polymorphism.A total of 69859 single genes were obtained from the mantle transcriptome of Perna viridis.Furthermore,a total of 378 shell proteins from Perna viridis were identified by proteomics/transcriptome method,including 132 SMPs with more than two peptide segments.Among 132 kinds of SMPs,there are 69 kinds of nacre,12 kinds of myoprismatic,and 51 kinds of them.WLP and TLP-1 genes of Mytilus coruscus were highly expressed in the posterior obturator muscle and mantle.The recombinant expression of WLP and TLP-1 played an important role in inhibiting the morphology,polymorph,binding capacity and crystallization rate of calcium carbonate crystal in vitro.The molecular localization of WLP and TLP-1 in the outer membrane,posterior obturator and prismatic layer of mussel was analyzed by immunohistochemistry and immunofluorescence analysis.The results show that WLP and TLP-1 have multiple functions in biomineralization and the interface between posterior obturator and shell.The results of pull-down experiment show that there are many protein networks interacting with WLP and TLP-1 in the shell.On the one hand,the above research has obtained systematic research data from the structural composition of different micro-levels of Perna viridis shells,the transcriptome of the outer membrane tissue,and the proteomics of Perna viridis shells,and it is the subsequent regulatory effect of shell matrix proteins on different structural levels of shells The mechanism laid the foundation.On the other hand,the above studies provided clues to explore the biomineralization mechanism of mussel shells and also laid the foundation for subsequent protein engineering research based on WLP and TLP-1.