| Apostichopus japonicus as a model organism for regeneration,when encountering adverse environmental conditions or predators,it has a special defense mechanism of "evisceration ".after the environmental conditions improve,it can quickly regenerate lost internal organs,which has aroused the interest of many researchers at home and abroad,and Apostichopus japonicus also become an important model for the study of organ regeneration in Echinodermata.As a transmembrane proteoglycan,SDC interacts with a large number of ligands(such as growth factors,adhesion receptors,soluble small molecules,proteases and extracellular matrix proteins),thus initiating many biological events related to cell adhesion,angiogenesis,inflammation and tissue repair.Studies have shown the SDC plays an important role in damage repair and regeneration.We found that th e related enzymes and components of ECM in intestinal regeneration of Apostichopus japonicus were changed,but how the above genes mediate the regulation of regeneration signal,whether SDC is involved in and the SDC expression mode are not clear.In this study,we obtained the EST fragment and the 3’UTR of the sdc gene,a total of 1100 bp.SDC has a largest open reading frame of 813 bp and 3’UTR of 259 bp,open reading frame encodes 270 amino acids,after predicting.It mainly contains SEA,single transmembrane region and 4.1m conserved domain.The 4.1m domain can be used as a CD domain in sdc4.Under certain conditions,when the conformation of the protein changes,it can participate in ECM adhesion and realize transmembrane signal transduction.Similar to all SDCs,Aj-SDC also has a single transmembrane domain composed of 23 amino acids.Sea domain is known to assist or regulate the binding with carbohydrate,In Aj-SDC,this domain constitutes ed domain,which provides the attachment site of HS or CS chain and may participate in the cell adhesion process.The predicted molecular weight is 30.08 k Da,it is a hydrophilic protein,its isoelectric point is 4.2.The NJ phylogenetic tree of the SDC protein of sea cucumber Apostichopus japonicus and other 37 representative species was obtained.The results showed that in vertebrates,different types of SDC were clustered into a large vertebrate evolutionary branch,while other invertebrates were clustered into a large branch.In the phylogenetic tree,the closest relative of sea urchin to sea cucumber was purple sea urchin.The two of them clustered together to form a small evolutionary branch.The branch of the phylogenetic tree was in line with the evolutionary status of the species.After prokaryotic expression,Aj-SDC was a soluble protein,and purified for further functional study.The results of real-time PCR showed that at the transcriptional level of sdc increased first and then decreased,reaching the peak on the third day after the evisceration.Though Western blot method showed that the expression trend of SDC translation level and transcription level were also showed this trend.It shows that the translation level of SDC is very similar with transcriptional expression.Through immunofluorescence experiments,it can be determined that SDC protein is widely expressed in various tissues of the regenerated intestine of sea cucumber,and expressed strongly in the mesentery.By analyzing the expression pattern of SDC,we speculate that SDC may be involved in the regeneration process of sea cucumbers.It may be combined with regeneration related factors or ECM outside the cell or on the cell membrane,there by participating in the regulation of cell adhesion,cell proliferation,migration and other processes during intestinal regeneration. |
