Functional Analysis Of Caspase Family Genes Of Dugesia Japonica

Apoptosis is one of the autonomous programmed deaths of organisms and is the normal physiological function of organisms.Regeneration mainly refers to the process of reconstruction and recovery after the damage of the tissue or organ of organisms.Dugesia japonica has strong regenerative capacity,but the role of apoptosis in the regeneration and the relationship between the apoptosis and regeneration are rarely reported.In this study,Dugesia japonica was used as experimental material.Four apoptotic genes DjCasp3 a,DjCasp3b,DjCasp3 c and DjCasp8 a were selected from the transcriptome database,Their encoded protein was analyzed by bioinformatics;the function of 4 genes in planarian were studied by Real-time PCR technology,RNAi technology,TUNEL staining and H3 P immunofluorescence.The results of bioinformatics analysis indicated that the apoptotic proteins encoded by DjCasp3 a and DjCasp3 c contained the conserved pentapeptide sequence QACRG,and the pentapeptide sequences encoded by DjCasp3 b and DjCasp8 a were QACKG and DACRG,respectively.The four gene-encoded proteins are all hydrophilic stable proteins;there are no transmembrane structures and secretory signal peptides,so they are not part of membrane receptor proteins and secreted proteins;subcellular localization shows that they are all located in the cytoplasm.The results of Real-time PCR showed that the four genes were dynamically expressed during the regeneration.The expression of DjCasp3 a,DjCasp3b and DjCasp3 c was increased extremely significantly 2-5 times at 3 hours and 3-10 times higher at 72 hours after amputation.DjCasp8 a was increased extremely significantly at 3 hours and 72 hours after amputation,but no higher expression than the previous three genes.The results of in situ hybridization showed that the 4 genes were widely expressed in the planarian,mainly in the parenchymal tissues on both sides of the body.The results of 4 gene RNAi showed that 4/20 of the whole planarians showed head damage after DjCasp3 a RNA interference,and 2/20 of the whole planarians showed head damage after DjCasp3 b RNA interference,but the planarians did not die,and gradually recovered after a period of time.There was no significant phenotypic change in the regenerative planarian.After DjCasp3 c and DjCasp8 a RNA interference,there was no obvious phenotypic change in either the whole or the regenerative planarians.The results of TUNEL staining showed that the TUNEL positive cells of the whole planarian were distributed in a scattered manner.The apoptotic cells were mainly concentrated in the wound within 150 ?m at 3 hours after amputation.The TUNEL positive cells were distributed systemically at 72 hours after amputation.After 4 gene interference,the number of TUNEL positive cells was significantly reduced in whole planarian.and the number of TUNEL positive cells decreased significantly at 72 hours after amputation.The results of H3 P immunofluorescence showed that the number of H3 P positive cells was significantly reduced in whole planarian afer 4 genes interference respectively.The number of H3 P positive cells was significantly reduced in the 3 hours after amputation respectively.but the number of H3 P positive cells did not change significantly compared with the control at 72 hours after amputation respectively.In summary,the results indicate that the 4 gene regulates apoptosis and stem cell proliferation in the planarian,which plays an important role in maintaining the homeostasis of the planarian's head tissue.