Bcl-xl, Bax2, and NF-κB are well-known to be involved in anti-apoptosis response. Although Bcl-xl has been reported in fish, the NF-κB-mediated regulatory mechanism and anti-apoptotic function are still unclear. Here, we cloned and characterized the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) Bcl-xl (CiBcl-xl) and its promoter region sequence. The full-length cDNA 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 shared high homology with Dario rerio Bcl-xl (DrBcl-xl). After stimulation with Poly I:C, the expression of CiBcl-xl in CIK cells and various tested tissues of grass carp were significantly upregulated. To further understand the transcriptional control of fish Bcl-xl induced by NF-κB, CiC-rel and Cip65 were expressed in Escherichia 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 CiBcl-xl promoter. Dual-luciferase reporter assays showed that CiC-rel and Cip65 activated CiBcl-xl promoter. Also, knockdown of CiC-rel and Cip65 reduced the expression of Bcl-xl. Therefore, similar to those of mammals, fish C-rel and p65 can upregulate the transcription of Bcl-xl. In addition, we found that overexpression of CiBcl-xl in CIK cells increased the cell activity and inhibited cell apoptosis, while overexpression of Bax2 promoted cell apoptosis. Meanwhile, co-transfection of CiBcl-xl and CiBax2 into cells can ease up apoptotic rate. To further investigate the molecular basis of synergistic effect of Bcl-xl and Bax2, we showed that Bcl-xl and Bax2 interacted with each other. The results suggested that Bcl-xl executed its anti-apoptotic function by binding to and inhibiting the pro-apoptotic activity of Bax2.

众所周知，Bcl-x1，Bax2和NF-κB参与抗凋亡反应。尽管在鱼类中已经报道了Bcl-xl，但是NF-κB介导的调节机制和抗凋亡功能仍不清楚。在这里，我们克隆并表征了草鱼（Ctenopharyngodon idella）Bcl-xl（CiBcl-xl）的全长cDNA序列及其启动子区域序列。CiBcl-x1的全长cDNA为2836 bp，ORF为627 bp，编码208个氨基酸的多肽。系统进化树分析表明，CiBcl-xl与Dario rerio Bcl-xl（DrBcl-xl）具有高度同源性。用Poly I：C刺激后，CiBcl-xl的表达CIK细胞中的草甘膦和草鱼的各种测试组织均显着上调。为了进一步了解由NF-κB诱导的鱼Bcl-xl的转录控制，CiC-rel和Cip65在大肠杆菌BL21中表达，并通过Ni-NTA His-Bind树脂进行亲和层析纯化。在体外，凝胶迁移率变动分析显示出的高亲和力次C-rel和次P65与CiBcl-XL启动子。双重荧光素酶报告基因测定表明Ci C-rel和Ci p65激活了CiBcl-xl启动子。另外，Ci C-rel和Ci的组合p65降低Bcl-xl的表达。因此，类似于哺乳动物的那些，鱼C-rel和p65可以上调Bcl-xl的转录。另外，我们发现CIK细胞中Ci Bcl-x1的过表达增加了细胞活性并抑制了细胞凋亡，而Bax2的过表达促进了细胞凋亡。同时，将CiBcl-1和CiBax2共转染到细胞中可以减轻细胞凋亡率。为了进一步研究Bcl-x1和Bax2协同作用的分子基础，我们证明了Bcl-x1和Bax2相互作用。结果表明，Bcl-xl通过结合并抑制Bax2的促凋亡活性来执行其抗凋亡功能。