Grass Carp (Ctenopharyngodon Idella) Bcl-xl:Transcriptional Regulation And Antiapoptosis Analysis

Bcl-xl,Bax2 and NF-?B are well-known to be involved in anti-apoptosis response.Although Bcl-xl has been reported in fish,the NF-?B-mediated regulatory mechanism and anti-apoptotic function is still unclear.In the current study,the grass carp(Ctenopharyngodon idella)Bcl-xl ORF and its promoter sequence were cloned and identified.The cDNA sequence of CiBcl-xl is 2836 bp with an ORF of 627 bp encoding a polypeptide of 208 amino acids.Phylogenetic tree analysis revealed that CiBcl-xl shares high homology with Dario rerio Bcl-xl(DrBcl-xl).After stimulation with poly I:C,the expression of CiBcl-xl was significantly up-regulated in CIK(C.idellus kidney)cells and tissues.It indicates that CiBcl-x1 plays an important role in the antiviral immune mechanism.To further understand the transcriptional control of fish Bcl-xl induced by NF-?B,CiC-rel and Cip65 were expressed in E.coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin.In vitro,gel mobility shift assays demonstrated the high affinity of CiC-rel and Cip65 with Ci Bcl-xl promoter.Dual-luciferase reporter assays showed that CiC-rel and Cip65 activated CiBcl-xl promoter separately.Therefore,similar to those of mammals,fish C-rel and p65 can up-regulate the transcription of Bcl-xl.In contrast,knockdown of fish C-rel or p65 reduced its expression.In addition,We also found that over-expression of CiBcl-xl in C.idellus kidney cells(CIK)increased cell viability and resistance to apoptosis,while over-expression of Bax2 can promote cell apoptosis.Meanwhile,co-transfection of CiBcl-xl and CiBax2 in cells can ease up apoptotic rate.To further investigate the molecular basis of synergistic effect of Bcl-xl and Bax2,we performed a GST-Pull down analysis and showed that Bcl-xl and Bax2 can interact with each other.Our results indicated that the fish pro-survival Bcl-xl is a downstream anti-apoptosis gene in NF-?B pathway.This provides a reference for us to study the mechanism of apoptosis of grass carp in more depth.