Investigation of the Possible Role of RAD9 in Post-Diapaused Embryonic Development of the Brine Shrimp Artemia sinica

Abstract

Background: The cell cycle checkpoint protein RAD9 is a vital cell cycle regulator in eukaryotic cells. RAD9 is involved in diverse cellular functions by oligomer or monomer. However, the specific mechanism of its activity remains unknown in crustaceans, especially in embryonic diapause resumption of the brine shrimp Artemia sinica. Methods and Results: In the present article, a 1238 bp full-length cDNA of As–RAD9 gene, encoding 376 amino acids, was obtained from A. sinica. The expression pattern of As–RAD9 was analyzed by qPCR and Western blot. The mRNA expression level climbs to the top at the 10 h stage of embryo development, while the protein expression pattern is generally consistent with qPCR results. Moreover, the As–RADd9 related signaling proteins, As–RAD1, As–HUS1, As–RAD17, and As–CHK1, were also detected. Immunofluorescence assay showed that the location of As–RAD9 did not show tissue or organ specificity, and the intracellular expression was concentrated in the cytoplasm more than in the nucleus. We also explored the amount of As–RAD9 under the stresses of cold and high salinity, and the results indicate that As–RAD9 is a stress-related factor, though the mechanisms may be different in response to different stresses. Knocking down of the As–RAD9 gene led to embryonic development delay in A. sinica. Conclusions: All these results reveal that As–RAD9 is necessary for post-diapaused embryonic development in A. sinica.