The Role Of RAD9 In Embryo Restart Of Atemia Sinica

In the process of Artemia sinica embryonic development,changes in the external environment can lead to diapause in Artemia sinica embryos.During embryo diapause and reactivation,on the one hand the embryo needs to repair its DNA damage,and on the other hand the cell cycle to regulate the embry's reactivation process.When exogenous or endogenous DNA damage factors attack cells,the cell cycle checkpoint control protein RAD9 can participate in the cell cycle arrest,and at the same time,the damaged DNA in the cell is repaired.Restore the normal functioning of the cell cycle to ensure that diapause embryos develop normally.RAD9 is also involved in the regulation of transcription of downstream target genes,affecting nucleotide synthesis,induction of apoptosis,and embryonic development.At present,the role of RAD9 in Artemia diapause and early embryos has not been studied.Therefore,this study has important theoretical significance for further analysis of the regulation of RAD9 on cell cycle during the diapause of Artemia.In this study,the full length of As-rad9 gene of Artemia sinica was cloned for the first time.Real-time quantitative PCR was used as a means to detect the expression level and expression pattern of As-rad9 gene in the development of Artemia sinica during different periods of temperature stress and salinity stress.Western blotting was used to detect the expression patterns and expression levels of RAD9,RAD17,HUS1,RAD1,CHK1 and CDC25 A proteins during the development of Artemia,and small interfering RNA(siRNA)was used to determine RAD9 in Artemia.The role of early embryo development.The results showed that the As-rad9 gene is 1238 bp in length and encodes 377 amino acids.The protein has a molecular weight of about 42.6 kDa and a theoretical isoelectric point of about 6.48.It is a hydrophilic protein and is most likely localized in the nucleus by subcellular localization.The real-time quantitative PCR results showed that the As-rad9 gene showed the highest expression level in the 5 hours of embryonic development,and then gradually decreased to a gradual upward trend after 40 hours.The results of Western blot showed that the expression trend of RAD9 protein and mRNA expression trend were general.Consistently,it was also found that the protein expression levels of As-RAD9,As-RAD1,As-RAD17,As-CHK1 and As-CDC25 A were consistent with their expression levels at different embryonic stages.The results indicate that As-RAD9 is a stress-related factor that promotes or inhibits the expression of genes associated with cell cycle checkpoints during the reactivation of diapause embryos.It can be seen from the immunofluorescence experiment that RAD9 is expressed in every stage of Artemia sinica,and has no organ-organ specificity,and is mostly located in the nucleus.Small RNA interference experiments have shown that knocking out the As-rad9 gene increases the mortality of Artemia and delays the development of Artemia.This study analyzed the expression pattern,expression site and regulation of RAD9 during the restart of Artemia sinica,and demonstrated that RAD9 plays an important role in the restart of Artemia diapause embryos.These findings provide a reference for further understanding of the molecular mechanisms of diapause and elimination of Artemia.