Cloning,Expression And The Transcriptional Level Of Grass Carp(Ctenopharyngodon Idella) CHOP

CHOP(also called as DDIT3/GADD153)encodes a ubiquitous transcription factor related to the CCAAT/enhancer-binding protein(C/EBP)family.It is one of most important components in the network of stress-inducible transcription.Here,for the first time,we cloned and identified the full-length cDNA(KX013389)of grass carp(Ctenopharyngodon idella)CHOP by homology cloning strategy.CiCHOP is1520 bp in length,comprising 246 nucleotides of 5?-untranslated sequence,the longest open reading frame of 747-nucleotide and 527 nucleotides of 3?-untranslated sequence.The longest open reading frame of CiCHOP translates into 248 aa with a typical DNA binding domain(BRLZ domain)and shares significant homology to the known CHOP counterparts.Phylogenetic tree analysis revealed a closer evolutionary relationship among fish counterparts,especially with Danio rerio CHOP.ATF4 is a central regulator which regulates the adaptation of cells to ER stress.Treatment with(TM),both CiCHOP and CiATF4 were significantly up-regulated.In order to know more about the role of CiCHOP under TM induction,in this study,we obtained the purified CiATF6N(CiATF6-nBLRZ)and CiATF4(CiATF4-nBLRZ)by affinity chromatography with the Ni-NTA His-Bind resin,and cloned the promoter sequence of CiCHOP.Gel mobility shift assays were employed to analyze the interaction of CiATF4 with the promoter of grass carp CHOP.The result showed that CiATF4 bound to the promoter of CHOP with high affinity by means of its BRLZ mainly.To further study the transcriptional regulatory mechanism of CiCHOP,recombinant plasmids of pGL3-CHOP was constructed and transiently co-transfected with pcDNA3.1-CiATF4 into C.idella kidney(CIK)cells.The result showed that the over-expression of ATF4 is essential for the transcriptional activation of CHOP.In addition,over-expression of CiCHOP in CIK cells facilitates the decrease of cell number.