The Role Of TRPM5 In Diapause Embryo Restart Of Artemia Sinica

Artemia sinica is a kind of low aquatic crustacean living in the environment of wide temperature and high salt tolerance.It has the advantages of resisting the adverse environmental conditions,short growth cycle and high nutritional value.It is also an open food for aquatic animals such as fish,shrimp and crab,and also an experimental material for studying cell biology,developmental biology and molecular biology.When severe environmental conditions such as hypoxia,low temperature,and dryness occur,female Artemia will produce diapause embryos wrapped in a hard shell.This embryo will enter the stage of developmental pause.When the environment is suitable,the embryo will restart,break the diapause state and develop into nauplius.TRPM5 is the fifth member of transient receptor potential cation channel subfamily M,which exists in many tissue types.It is a calcium activated cation channel and mediates plasma membrane depolarization.TRPM protein family plays an important role in cell cycle regulation,apoptosis and other biological processes,and has the characteristics of temperature sensitivity.Most of its members are related to cold stimulation signal transduction.Artemia need cold stimulation process such as freezing in the process of breaking diapause.Therefore,As-trpm5 is likely to be closely related to the process of low-temperature diapause and restart of Artemia sinica embryos.At present,the role of As-trpm5 gene in the process of diapause embryo restart has not been reported.In this study,a 2211 bp trpm5 gene fragment of Artemia sinica was cloned for the first time and analyzed by bioinformatics software.Real time-q PCR was used to detect the m RNA expression of trpm5 gene in Artemia sinica at different developmental stages and under low temperature and high salt stress.The protein expression levels of As-TRPM5,As-CIRBP,As-CIB1 and As-CEBPA at different developmental stages and under low temperature and high salt stress were detected by Western Blot,and the expression sites of As-TRPM5 were detected by immunofluorescence technique.The results showed that the obtained sequence contained 1791 bp open reading frame(ORF)and predicted 596 amino acids with a theoretical isoelectric point of 5.35.Bioinformatics analysis predicted that As-TRPM5 protein was hydrophilic and contained a transmembrane region.According to the results of Real time-q PCR,the expression trend of As-trpm5 gene in the embryonic development of Artemia sinica was high at first and then low,and reached the highest level at 10 h.Western blot showed that the expression trend of As-TRPM5 protein and its m RNA was consistent.In the study of low temperature and high salt stress,Real time-q PCR and Western blot showed that As-TRPM5 was highly expressed at low temperature,but lower than the control group at high salinity.Western blot results of As-CIRBP,As-CIB1 and As-CEBPA proteins related to As-TRPM5 showed similar expression trend with that of As-TRPM5 under corresponding conditions.Immunofluorescence results showed that As-TRPM5 protein was not organ specific and expressed in different stages of Artemia sinica.It was located in the cell membrane and cytoplasm.The results show that As-TRPM5 plays an important role in the process of releasing diapause embryos and restarting of Artemia sinica at low temperature.It is of great theoretical significance to expound the molecular regulation mechanism of the development and restarting of Artemia sinica diapause embryos,and has potential value for the development of Artemia sinica resources.