The Role Of Checkpoint Kinase RAD17 In The Diapause Process Of Artemia Sinica

When Artemia sinica is in harsh climate conditions(such as high salt and low temperature environment),its embryo can stop its own development and enter the diapause stage.Most diapause embryos stop developing in the gastrula stage of halogen cells,and form unique diapause eggs to resist the damage caused by adverse environment to the embryo,and break the diapause to continue to grow under the appropriate growth environment.RAD17 is involved in a variety of physiological and metabolic activities related to cell cycle regulation,such as DNA damage repair,promoting programmed cell apoptosis and cell cycle arrest.Up to now,the mechanism of rad17 in diapause and diapause removal of Artemia sinica in China is still unclear.In this experiment,the full length sequence of rad17 gene of Artemia sinica was cloned for the first time using RACE technology.As-rad17 is 1,900 bp in length and contains an open reading frame(ORF)of 1632 bp,a 5’-utr of 156 bp and a 3’-utr of 202 bp.It encodes 543 amino acid proteins,with theoretical molecular weight of about60.7kDa and theoretical isoelectric point of 5.20.The software analysis shows that RAD17 protein is a hydrophilic protein located in the nucleus and cytoplasm.No transmembrane region,is a non-transmembrane protein without secretion;Phosphorylation site predictions suggest that there may be 24 serine sites,8 threonine sites,and 12 tyrosine sites in Chinese Artemia As-rad17.The prediction results of secondary structure showed that As-rad17 was dominated by helix(43.46%),with extended chain(17.68%),fold(7.92%)and random curl(30.94%).By constructing the phylogenetic tree of as-rad17 protein,we analyzed and compared the homologous relationship between Artemia sinica and 13 other species,and confirmed that as-rad17 protein was highly conserved.The results of in situ hybridization showed that As-rad17 has almost non-specific expression in the whole body of Artemia.The double experiments of Real-time PCR and western blotting showed that the expression level of RAD17 first increased and then decreased in the early development stage of Artemia sinica embryo development at 0-40 h,and the expression level was higher at 15-20 hours.In the process of stress experiments,we found that the expression level of as-rad17 showed a different trend under the stimulation of low temperature and high salt.The expression level of as-rad17 was up-regulated under low temperature stress,while the expression level of as-rad17 was slightly down-regulated under high salt stress with no obvious change.The overall experimental results show that RAD17 is involved in the regulation of diapause and diapause release in the early embryo of Artemia sinica in China,and plays a crucial role in the above metabolic processes.