Research On Senescence-related Genes FoxO,Akt,Sir2 Based On The Reproductive Transformation Of Daphnia Pulex

Daphnia pulex is a freshwater miniature crustacean that is distributed in inland waters all over the world.FoxO is a forkhead box transcription factor,which regulates cell survival and senescence through processes such as transcriptional regulation and signal transduction.Akt is a serine/threonine protein kinase that plays an important role in cell growth,survival and lifespan regulation.Sir2 is a deacetylase,which is closely related to lifespan regulation.In this paper,we used D.pulex as a research object.The full-length c DNA of FoxO was obtained from D.pulex.The expression of FoxO、Akt 和 Sir2 mRNA and protein was detected by q PCR and western blot.Continuous RNA interference was performed on the FoxO and Akt genes of D.pulex,and the mRNA and protein expressions of FoxO、Akt and Sir2 related to aging before and after the interference were detected.Preliminarily explore the role of FoxO、Akt and Sir2 genes in the senescence and reproductive transformation of D.pulex.The results obtained in this paper are as follows:(1)The full-length c DNA of FoxO gene in D.pulex was cloned by RACE(Gen Bank No.MN412521),containing 2121 bp open reading frame(ORF),encoding 706 amino acids.FOXO protein has a highly conserved Forkhead domain and transcriptional activation domain.The amino acid sequence of FOXO protein in D.pulex was 77% similar to that of Daphnia magna.Discovered from the phylogenetic tree,FOXO of D.pulex has a close relationship with D.magna.On the contrary,it was far related to Homo sapiens,Mus musculus.(2)The FOXO antibody was obtained by the whole gene synthesis method.The mRNA and protein expressions of FoxO,Akt and Sir2 of different ages were detected.The results show that the expression of FoxO、Akt and Sir2 in D.pulex has a period difference,they were expressed at a higher level in senescent D.pulex.This is due to the combination of genes with the promoter region of target genes,which enhances the clearance of peroxides and the resistance of cells to oxidative stress.Moreover,the expression levels of these three genes were high at 15-20 d.It is speculated that 15-20 d is a critical period for D.pulex to enter senescence under 25℃ culture conditions.(3)RNA interference was performed on the FoxO gene of D.pulex.The results showed that the expression of FoxO after interference was significantly lower than that before interference(P<0.05),and the expression trend after interference was similar to that before interference.The study found that after reducing FoxO expression,the mRNA and protein expression of upstream gene Akt and downstream gene Sir2 were significantly decreased(P<0.05).It shows that FoxO,Akt and Sir2 have a positive regulation relationship.Compared with the negative control group and the blank control group,the interfering group D.pulex appeared egg saddles at 27 days.It shows that the interference can lead to the reproductive conversion of D.pulex in advance.(4)Continuous RNA interference was performed on the Akt gene of D.pulex.The results showed that Akt mRNA and protein expression after interference were significantly lower than before interference(P<0.05),and the average interference efficiency could reach 88%.It was found that after feeding bacteria containing Akt＿ds RNA the mRNA and protein expression of FoxO and Sir2 decreased significantly(P<0.05).These results indicate that FoxO and Akt have a positive regulatory relationship,and FoxO,Akt and Sir2 work together to regulate aging.Compared with the negative and blank control groups,egg saddles appeared in the Akt interference group at 28 days,indicating that interference with the Akt gene can lead to the reproductive conversion of D.pulex in advance.