| Pteria penguin is a large marine economic shellfish that produces mabé pearls and relies on strong byssus to firmly attach itself to a hard substrate,resist the impact of currents,and predation.The byssus was composed of byssal proteins secreted by the foot glands,and there are three main types of byssal proteins: one constitutes the byssal cuticle,one constitutes the byssal core,and the other constitutes the adhesive plaque.However,studies on P.penguin foot gland,byssal protein composition and byssogenesis mechanism have not been reported.Therefore,this paper aims to investigate P.penguin foot gland species by histological methods.The proteomic approach was used to investigate byssal protein composition and to analyze the important proteins involved in the process of byssus secretion.The transcriptomic approach was used to investigate the similarities and differences in the byssogenesis mechanisms of P.penguin during the winter and summer seasons.The main studies and results are as follows:1.Paraffin sections and ultrathin sections were made of foot with byssus(FW)and foot without byssus(FO)in this experiment.Sections were stained by masson′s trichome staining and sirius red staining,observation by light microscope and polarized light microscopy(PLM),combined with TEM and SEM,which revealed the presence of collagen glands and phenol glands that secreted byssal proteins.P.penguin byssal proteins were transported by vesicles in both glands,and the secretory granules in the vesicles were divided into three categories: oval secretory granules of collagen gland,subrounded secretory granules with high electron density of phenol gland,and loosely distributed positional secretory granules with variable density.Transparent adhesive material was present on the surface of the foot,but no mucus gland-related structures were found in the foot.SEM observation of the byssus revealed a flat surface,the presence of granules embedded in a protein matrix,and a neatly arranged and tightly packed thread-like fiber inside.There was no obvious junction between the byssal thread and the end of the byssus,and the surface structure of both was consistent,but the contact area between the end of the byssus and the substrate showed a foam-like structure.The results of this study help to understand the structure of the foot glands and byssus of P.penguin.2.4D lable free quantitative proteomics analysis was performed on P.penguin feet(FW and FO),byssus and pearl pocket(PP).48422 peptides belonging to 3721 proteins were detected and quantified.1105,1049 and 157 differentially expressed proteins(DEPs)were identified in three comparison groups,FW vs PP,FO vs PP and FW vs FO,respectively.Compared with FO,the DEPs in FW were enriched in vesicle secretion-related and microtubule-related proteins.Combined with the TEM observation of FW,it was found that byssal precursor protein was transported to the ventral groove through vesicle transport and cilia movement and microtubule protein during byssus secretion process,and then self-assembled into byssus for adhesion.Tyrosinase capable of modifying DOPA were found in both the P.penguin foot and byssus.The differentially expressed proteins in the foot were mainly located in the extracellular region and 7 of the31 proteins in the byssus were derived from the extracellular matrix(ECM),and protease inhibitors and CD109 antigens associated with byssus self-defense were present in the proteins of byssus.The results of this study have contributed to the understanding of the process of byssus secretion in P.penguin and have enriched the study of the protein composition of byssus in bivalves.3.Transcriptome sequencing combined with polyphenol oxidase(PPO)activity and reactive oxygen species(ROS)assays were performed on the foot with byssus(WB)and foot without byssus(NB)at summer(27 ℃)and winter temperatures(21 ℃)in a simulated P.penguin habitat to investigate the possible mechanisms of byssus formation.The results showed that PPO is an important enzyme in byssus formation,and the PPO activity was higher in summer than in winter without significant difference.The ROS in the foot was also higher in summer than in winter,but there was no significant difference.A total of 28442 genes were obtained by transcriptome analysis,and 1100 and 2573 genes were identified as differentially expressed genes(DEGs)in the two comparison groups WB21 vs NB21 and WB27 vs NB27,respectively.GO and KEGG enrichment analysis of the two comparison groups revealed that differentially expressed genes were enriched in both endoplasmic reticulum(ER)and protein processing in the endoplasmic reticulum associated with correctly-folding,with both up-regulated and down-regulated of HSP70,and ubiquitin-protein ligase genes(E2,E3 ubiquitin-protein ligases)also showing up-regulation.In addition,microtubule-related genes were significantly enriched in the27 ℃ group,kinesin and dynein-like gene were significantly upregulated in it,which was consistent with the proteomic results.The expression of protease inhibitor-related genes appeared to be up-regulated in both comparison groups,which was also consistent with the proteomic results.The results of the study provide a preliminary basis for understanding the possible mechanisms of byssus secretion in P.penguin. |
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