The Role Of As-MBD2 In The Diapause Embryo Development Process Of Artemia Sinica

Methylated CpG-binding domain proteins(MBDs)play an important role in epigenetic regulation.MBD family contains Me CP2,MBD1,MBD2,MBD3,MBD4,MBD5 and MBD6,all of which contain a common structural domain MBD.mbd2 can bind to CpG islands or methylated DNA in promoters and recruit histone deacetylases and DNA methyltransferases to perform gene regulation functions and promote gene expression.Alternatively,mbd2 can function as a transcriptional repressor in gene silencing or as a scaffolding protein targeting the GATAD2 A and GARTAD2 B structures of chromatin to repress downstream gene transcription.In addition,mbd2 can also enhance the expression of unmethylated c AMP molecules to activate promoters and selectively repress the transcriptional activity of methylated r RNA promoters.In Artemia sinica,embryos are stalled when stressed by adverse environmental conditions such as low temperature,high salinity and altered photoperiod,resulting in stalled eggs.When the external conditions are suitable,the stagnant embryos can break the stagnation period and restart embryonic development.Previous studies have revealed that MBD2 plays an important role in the development of stalled embryos,but its molecular regulation mechanism is still unclear and needs to be further investigated.In this paper,a 1469 bp m RNA fragment of As-mbd2 gene was obtained by RACE(rapid amplification of c DNA ends)for the first time.The expression site of As-mbd2 in Artemia sinica was analyzed by immunofluorescence assay,and it was found to be widely expressed and present in the nucleus,especially in Artemia sinica.Real-time q PCR and immunoblotting revealed that the relative expression of As-mbd2 was highest at 15 h,but no significant changes occurred at other stages.The expression of As-MBD2 was significantly higher at low temperatures and during adverse environmental stresses such as high salinity,suggesting an important role in the re-initiation of lagging embryos and early embryonic development in Artemia sinica.We also examined the expression of As-CDK2,As-CDK6,As-Cyclin E and As-SETD4,which are related to the regulation of As-MBD2,during embryonic development of Artemia sinica,by Western Blot technique.The experimental results showed that As-MBD2 and the four related proteins,CDK2,CDK6,Cyclin E and SETD4,were expressed in the same way as As-MBD2 in the developing embryo of Artemia sinica,and their expression patterns and mutual regulation were preliminarily analyzed,indicating that As-MBD2 and these four proteins promoted the development of Artemia sinica embryos.The experimental results of this study provide a basis for further understanding the molecular regulation mechanism of hysteretic embryo restart in Artemia sinica,and provide a basis for further research on the regulatory role of As-MBD2 in the hysteretic process of other arthropods,which is of great theoretical significance.It has potential applications in the development and utilization of the Artemia sinica embryo resources.