The Role Of As-TIM2 In The Reactivation And Early Embryonic Development Of Artemia Sinica Diapause Embryos

Artemia sinica is a small crustacean and has a strong ability to resist harsh environmental conditions.When A.sinica is under harsh environment,it will produce diapause eggs.The formation of diapause eggs will inhibit all life activities,which can effectively resist the influence of the harsh environment.When the external environment is appropriate,then diapause can be broken and the embryo re-enters the development cycle.Due to this unique biological characteristic,the molecular mechanism of diapause production and release of diapause has not been completely elucidated,and it has become a hot issue in biological research.tim2(timeless2 or timeout)plays a vital role in chromosome stability and normal DNA metabolism.As a homolog of tim2 gene,tim1 is a recognized core biological clock gene.In addition to being closely related to embryonic development and diapause,in mammals,tim1 is an indispensable part of cell cycle control,DNA replication,DNA damage response(DDR),sister chromatid cohesion,early embryonic development and cell apoptosis.TIM2 and mammalian TIM1 are highly conserved at the protein level.The investigation of the function of mammalian TIM1 and related mechanisms has laid a good foundation for the study of the role of TIM2 in arthropods,and has important reference value.Therefore,this study plays an important role in analyzing the role of As-TIM2 in the reactivation of A.sinica diapause embryos and the development of early embryos,and has potential application value for the development of new animal proteinogens.In this study,the As-tim2 gene sequence is successfully cloned for the first time by using RACE technology,with a length of 3708 bp,encoding 1236 amino acids,and molecular weight is 143.4KDa.The expression level of As-TIM2 is analyzed by RT q PCR and WB.The expression level of As-TIM2 is basically similar to the gene level,that is,the expression level showed an exaltation from 0h to 5h.It reaches a peak before 10 h,and gradually declines with the development of A.sinica.The Western Blot experiment results showed that the expression trend of As-TIM2 is similar to that of As-PLK1,As-Aurora A,As-CDK1 and As-Cyclin B at different stages of embryonic development;In addition,the response of As-TIM2 and related proteins As-PLK1,As-Aurora A,As-CDK1 and As-Cyclin B under high salt and low temperature stress environment are analyzed,and the results show under high salt and low temperature stress,the expression of As-TIM2 shows a trend of high expression,while the expression of related proteins As-PLK1,As-Aurora A,As-CDK1 and As-Cyclin B show a trend of low level expression.The results of immunofluorescence show that As-TIM2 was expressed at all developmental stages of A.sinica.This study combines the expression levels of As-TIM2 and its related proteins at different developmental stages of A.sinica,the response under stress conditions and the subcellular localization analysis of As-TIM2 and As-PLK1,which proves that As-TIM2 plays an important role in the reactivation and early embryonic development of A.sinica diapause embryos.The in-depth study of arthropods and TIM1 in mammals provides a reference.