斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。  

IDENTIFICATION AND PROMOTER ANALYSIS OF AN IFIT FAMILY GENE FROM ZEBRAFISH DANIO RERIO

IFIT family consists of a group of interferon-induced proteins with Tetratricopeptide Repeat(TPR) motifs.Whereas there are four members in mammals,it is uncertain whether the same IFIT family members exist in fish and whether the fish homologues display the same expression pattern.Our previous report identified two genes from crucian carp Carassius auratus,which are similar to mammalian IFI58 and IFI56 and are induced by grass carp reovirus(GCRV) and interferon(IFN)-containing supernatant.However,unlike mammalian IFI56 gene,virus-induced expression of both genes is blocked by addition of cycloheximide(CHX),a potent inhibitor of protein synthesis.In order to further investigate whether there is an orthologue of mammalian IFI56 in zebrafish,we first searched zebrafish genome data by using mammalian IFI56 sequence.As expected,we identified a hypothetical zebrafish gene,named zebrafish IFIT-A gene,which contained a conserved gene structure similar to mammalian homologues.Zebrafish IFIT-A gene had the largest ORF that encoded a 429-amino-acid protein with conserved TPR motifs.Similar to mammalian IFI56,zebrafish IFIT-A gene was composed of an intron and two exons.RT-PCR showed that zebrafish IFIT-A mRNA was induced in ZFL cell by Poly I:C treatment.Sequence analysis of flanking region of zebrafish IFIT-A gene revealed a typical IFN-stimulated response element(ISRE),which was usually found in the promoters of Interferon Stimulated Genes(ISGs).Consistently,Poly I:C and recombinant crucian carp IFN protein were able to activate the activity of zebrafish IFIT-A promoter.Finally,overexpression of zebrafish IRF3 and IRF7 resulted in a significant increase in promoter activity of zebrafish IFIT-A gene.These data together provided evidence that zebrafish IFIT-A belonged to fish IFIT family and that fish IRF3 and IRF7 might play an important role in regulation of zebrafish IFIT-A expression.