大黄鱼HIF-1α基因的克隆鉴定与表达分析

MOLECULAR CHARACTERIZATION AND EXPRESSION ANALYSIS OF HIF-1αIN LARGE YELLOW CROAKER(LARIMICHTHYS CROCEA)

为研究低氧诱导因子1α(Hypoxia-inducible factor-1α,HIF-1α)在鱼类免疫应答中的作用,研究克隆得到了大黄鱼(Larimichthys crocea)HIF-1α基因(LcHIF-1α)的cDNA序列,其开放阅读框全长2256个核苷酸,编码一个751个氨基酸的蛋白。通过氨基酸序列比对发现,LcHIF-1α蛋白具有5个典型的功能结构域,包括1个helix-loophelix(HLH)结构域,2个PAS结构域,1个DNA结合结构域(HIF-1)和1个羧基端反式激活结构域(HIF-1a_CTAD)。系统进化分析显示,LcHIF-1α和其他硬骨鱼类HIF-1α聚成一支,与两栖类、鸟类和哺乳类HIF-1α的亲缘关系相对较远。序列分析结果表明,LcHIF-1α具有保守的特征结构域,预示着其功能可能与哺乳动物HIF-1α类似。荧光定量PCR结果显示,LcHIF-1α在所检测的健康大黄鱼各个组织中都有表达,在溶藻弧菌(Vibrio alginolyticus)感染后,大黄鱼脾脏和头肾组织中的LcHIF-1α表达水平显著上调,达到峰值时分别上升了6.8倍和2.9倍。此外,LcHIF-1α在大黄鱼的3种免疫细胞(中性粒细胞、巨噬细胞和淋巴细胞)中也都有表达,在中性粒细胞和巨噬细胞中的表达量相对较高;在脂多糖(LPS)诱导后,中性粒细胞和巨噬细胞中LcHIF-1α的表达量显著增加,峰值分别是对照组的2.6倍和1.8倍,这些结果表明LcHIF-1α可能参与调控大黄鱼抗细菌免疫应答。

Hypoxia-inducible factor-1α(HIF-1α)is considered as the master regulator of the response to hypoxia,through activating target gene expression involved in angiogenesis,erythropoiesis and glycolysis.In recent years,HIF-1αhas been found to regulate the immune response.However,very little information was available about its functions in fish immune response.In this study,the cDNA sequence of HIF-1αgene(LcHIF-1α)was cloned from large yellow croaker Larimichthys crocea.Its open reading frame(ORF)was 2256 nucleotides long,encoding a peptide of 751 amino acids.The deduced LcHIF-1αprotein contained five typical motifs:a helix-loop-helix(HLH)domain,two PAS domains,a DNA binding domain(HIF-1),and a HIF-1αC-terminal transactivation domain(HIF-1a_CTAD).Phylogenetic analysis showed LcHIF-1αformed a separate clade with sequences from other fish species,far from the clades formed by avian,amphibian and mammalian HIF-1αproteins.The LcHIF-1αtranscripts were constitutively expressed in all examined tissues and significantly up-regulated in the spleen and head kidney tissues by Vibrio alginolyticus,and reached the peak at 24h,with 6.8-and 2.9-fold increases,respectively.Its transcripts were also detected in neutrophils,macrophages and lymphocytes from large yellow croaker,and significantly up-regulated by lipopolysaccharide(LPS)in neutrophils and macrophages with 2.6-and 1.8-fold increases at the peak.These results indicate that LcHIF-1αmay play a role in mediating the immune response of large yellow croaker.