草鱼Bcl-2基因的克隆、组织表达分析及DHA诱导下其在脂肪细胞中的表达变化

Molecular cloning,tissue expression pattern of Bcl-2 in grass carp(Ctenopharyngodon idella)and its expression change after the treatment of DHA in in-vitro cultured adipocyte

为获知草鱼B细胞淋巴瘤-2(B cell CLL/lymphoma-2,Bcl-2)基因序列及其在草鱼各组织和脂肪细胞中的表达情况,采用cDNA快速末端扩增技术获得了草鱼Bcl-2的全长cDNA序列,通过实时定量PCR研究了Bcl-2在草鱼不同组织中的表达水平及经二十二碳六烯酸(docosahexaenoic acid,DHA)处理后,草鱼脂肪细胞Bcl-2的表达变化。结果显示,所获得草鱼Bcl-2基因全长cDNA序列长度为1 292 bp,包含133 bp的5'非翻译区和784 bp的3'非翻译区;开放阅读框为375 bp,编码124个氨基酸。草鱼Bcl-2与鲤、斑马鱼Bcl-2氨基酸同源性分别为89.5%和91.1%,与人、原鸡、小鼠、野猪、欧洲牛、褐家鼠、家猫和犬等其他动物的氨基酸同源性为57.7%～62.0%。其编码蛋白的分子量为14.14 ku,等电点为4.68。Bcl-2基因在草鱼心脏、肝胰脏、脾脏、鳃、肾脏、肌肉、腹腔脂肪组织、脑、肠和精巢10个组织中均有表达,其中在腹腔脂肪组织、肾脏和精巢中表达丰度最高,在肌肉中表达最低。DHA处理草鱼前体脂肪细胞后,Bcl-2基因表达在增殖阶段下调,而在分化阶段上调。研究表明,Bcl-2在草鱼各组织中广泛表达,且DHA对其在脂肪细胞中的表达具有调控作用。

To investigate the effects of docosahexaenoic acid （DHA）on the expression profiles of B cell CLL/lymphoma-2 （Bcl-2） in grass carp （ Ctenopharyngodon idella） different tissues and adipocyte in vitro, the rapid amplification of cDNA ends（RACE） technique was used to clone the full-length cDNA of Bcl-2 gene of grass carp, and the real-time PCR （qRT-PCR） was employed in the analysis of gene expression pattern in different tissues and in the in-vitro cultured adipocyte of grass carp. The results revealed that the full-length Bcl-2 cDNA is 1 292 bp and contains a 5＇-untranslated region with 133 bp and 3＇-untranslated region with 784 bp. The open reading frame （ORF）has 375 nucleotides encoding 124 amino acids. The deduced amino acid sequences of Bcl-2 of grass carp displayed higher similarities with Cyprinus carpio （89.5 % ）and Danio rerio（91.1% ） ,but lower identity to the other animals with the similarity ranging from 57.7% to 62.0%. The molecular weight of encoded protein was predicted at 14.14 ku with pI at 4.68. The Bcl-2 gene is widely expressed in heart, hepatopancreas, spleen, gills, kidney, muscle, intrapaneal fat body （ IPF）, brain, intestine and spermary. The highest expression level was observed in IPF, kidney, spermary and the lowest expression level was observed in muscle. The expression of other tissues was between IPF and muscle. After DHA treatment, the Bcl-2 expression was downregulated in the proliferation phase, but upregulated in the differentiation phase. This suggested the Bcl-2 was expressed widely, and DHA could regulate the expression of Bcl-2 gene.