Function Analysis Of Atg1 Family Gene In Dugesia Japonica

Dugesia japonica belongs to the genus Platyhelminthes,Turbellaria,Dugesiidae,and Dugesia.It plays a very important role in the evolution of animal systems.It has a strong ability to regenerate and has become a model animal for research in regenerative biology and developmental biology.In this expreiment,three homologous genes related to the autophagy-related gene Atg1 were screened from the Japanese worm transcriptome database:DjAtg1-1,DjAtg1-2 and DjAtg1-3.The functions of these three genes were analyzed by molecular biology techniques such as bioinformatics analysis,whole-mount in situ hybridization,RNA interferce,whole-mount immunostaining,quantitative real-time PCR and transmission electron microscopy(TEM).The results are as follows:(1)The DjAtg1-1 gene contains a maximum open reading frame(ORF),1458 bp in size,and encodes a protein composed of 485 amino acid polypeptides.The DjAtg1-2 gene contains a maximum open reading frame(ORF),833 bp in size,and encodes a protein composed of 243 amino acid polypeptides.The DjAtg1-3 gene contains a maximum open reading frame(ORF),2445 bp in size,and encodes a protein consisting of 814 amino acid polypeptides.The structure and evolutionary status of the deduced amino acids of DjAtg1-1,DjAtg1-2 and DjAtg1-3 were analyzed by bioinformatics,the results showed that the proteins encoded by DjAtg1-1,DjAtg1-2 and DjAtg1-3 all contain a serine/threonine protein kinase catalytic(S_TKc)domain,belonging to the Atg1 family of hydrophilic proteins.(2)The results of whole-mount in situ hybridization showed that the three genes of DjAtg1 were mainly distributed in intact worm.In regenerative worms,the hybridization signals were mainly distributed on both sides of the body and at the regenerative blastemas,and relatively strong on the 3~rdd day of regeneration.(3)After DjAtg1-1 and DjAtg1-2 interference,there was no significant change in morphology between the whole worms and the regenerative worms.At the same time,the results of Rt-PCR showed that the expression levels of DjAtg1-2,DjAtg1-3 and stem cell marker gene DjPiwiA were significantly up-regulated,and the expression levels of cell cycle regulatory genes DjcyclinA,DjcyclinB,Djcdk and neoblasts cell proliferation marker gene Djpcna were significantly down-regulated.After DjAtg1-3interference,the regeneration speed of the tail regenerative is slowed down,while the whole and tail regeneration head group had no significant change in morphology compared with the control group.At the same time,the results of Rt-PCR showed that the expression levels of DjAtg1-1,DjAtg1-2,DjpiwiA,DjcyclinA and DjcyclinB were significantly down-regulated.(4)whole-mount immunostaining showed:DjAtg1-1 and DjAtg1-2 were interfered,the number of H3P+cells in the experimental group was significantly increased compared with the control group;DjAtg1-3 was interfered,the number of H3P+cells in the experimental group was significantly reduced compared with the control group.(5)Transmission electron microscopy showed that DjAtg1-1 and DjAtg1-2 were interfered did not significantly affect the formation of autophages.Autophagy was inhibition after DjAtg1-3 gene interference.In conclusion,the three genes of DjAtg1 are involved in autophagy and stem cell proliferation.There may be complementary roles between DjAtg1 genes,while DjAtg1-3 controls the expression of DjAtg1-1and DjAtg1-2.DjAtg1-3 affects planarian regeneration and worm remodeling and plays an important role in the early stages of regeneration.