Expression Of As-CAV-1and As-SDH Protein During Embryonic Development、Low-temperature And High Salinity Stress In Artemia Sinica

Artemia mainly lives in the hypersaline waters, it belongs to Phylum Arthropoda, ClassCrustacea, Order Anostaca, Artemidae, Artemia. It and has a very strong ability resisting andadapting the extreme environment such as lower-temperature and high salinity. Meantime,because of the poor environment, Artemia produces dormant embryos for resisting badconditions.Caveolin-1mainly participates in regulating the intracellular signal transduction. WhileSorbitol dehydrogenase (SDH) mainly involved in polyol pathway. Both As-CAV-1andAs-SDH are playing an important role in embryonic diapause related pathway from Artemiasinica, but the interaction studies of the two proteins in Artemia sinica have not been reported.So, in this study, the interactions of the two proteins at the embryonic diapause from Artemiasinica are investigated by theoretical and experimental analysis.In this study, PCR technology was used to amplify the ORF fragments of As-cav-1andAs-sdh. The length ORF of As-cav-1is675bp, it encoded224amino acids, and the molecularweight is25KD; the length ORF of As-sdh is1047bp, it encoded348amino acids, and themolecular weight is37KD. Domain analysis revealed that As-CAV-1protein contains acaveolin superfamily, while As-SDH contains ADH_N catalytic domain and ADH_zinc_Ncoenzyme binding domain. The multiple sequence alignments and the construction ofphylogenetic tree of As-CAV-1and As-SDH were performed by software, and the resultsshowed that both of the two proteins were similar to invertebrate, the evolutionaryrelationships were mostly consistent with the classification status.The prokaryotic expression system was used for the expression of As-CAV-1andAs-SDH recombinant proteins. The two proteins were successfully expressed. Both of the twoproteins were purified with histidine affinity chromatography. Then, rabbit polyclonalanti-bodies were prepared with the purified recombinant proteins. The subcellular location ofAs-CAV-1was detected by immunofluorescence labeling method, the results showed thatAs-CAV-1was mostly expressed in cell membrane. The western bloting results of theexpression patterns about the two proteins under embryonic development、temperature andsalinity stress showed that As-CAV-1was highly expressed at0h, and dramatically decreased at40h, but incresed at3d. While As-SDH protein was lower expressed at0h, andup-regulated after0h until10h, then down-regulated until3d. At the temperature assay, theresults showed that As-CAV-1and As-SDH were both highly expressed at15℃. At thesalinity stress assay, the As-CAV-1and As-SDH were both increased at higher salinity stress.Our results suggested that both the As-CAV-1and As-SDH were all play a negativeregulation role in embryonic development and a synergistic effect role in the process ofenvironment stress, they were both participated in cell cycle, intracellular signaling andenergy metabolism. They were not only play an important role in embryonic development andenvironment stress but also the regulatory factor.