【摘要】 为探讨低等脊椎动物Foxp1与免疫细胞活化及机体雌激素水平的相关性,本研究首次分离克隆了尼罗罗非鱼Foxp1的开放阅读框序列,并通过Real-time PCR对其mRNA表达水平进行检测。结果显示,尼罗罗非鱼具有2种不同基因编码的Foxp1分子,分别命名为OnFoxp1a与OnFoxp1b;OnFoxp1a为1710 bp,由15个外显子编码569个氨基酸,OnFoxp1b为2040 bp,由16个外显子编码679个氨基酸;OnFoxp1a/b均含有Foxp亚家族的特征性结构,即锌指结构、亮氨酸拉链样结构和叉状螺旋结构;与OnFoxp1a相比,OnFoxp1b与高等脊椎动物Foxp1具有更近的亲缘关系。Real-time PCR检测结果显示,OnFoxp1a在精巢中有高水平表达,OnFoxp1b在心脏中有高水平表达,同时在免疫相关组织如鳃、脾脏、肾脏、肠等均有中等水平表达;淋巴细胞多克隆刺激剂PHA、PMA、LPS刺激尼罗罗非鱼外周血单个核细胞(PBMC),结果显示,50μg/mL PHA和50ng/mL PMA分别刺激6、12、24 h均显著增强OnFoxp1b的表达(P<0.05),20μg/m L LPS刺激后,OnFoxp1b的表达出现先降低后升高的趋势(P<0.05);而OnFoxp1a的表达除50μg/mL PHA刺激24h后有所升高,其余均无显著变化(P>0.05);雌激素处理6月龄雄性尼罗罗非鱼48h,OnFoxp1a/b在肠、肾脏中的表达显著上调(P<0.05),而脾脏中无显著变化(P>0.05)。综上所述,低等脊椎动物硬骨鱼类2种不同基因编码的Foxp1a/b均为哺乳动物Foxp1的同源分子,但其序列、结构特征、表达模式迥异,提示其功能发生歧化;同时两者的表达与淋巴细胞活化及机体雌激素水平密切相关。更多还原

【Abstract】 To investigate whether the mRNA expression of Nile tilapia( Oreochromis niloticus) Foxp1 a and Foxp1 b are involved in the lymphocyte activation and regulated by 17β-estradiol( E2) level in vivo,the coding sequences and mRNA expression patterns of On Foxp1 a and On Foxp1 b were characterized and analyzed by Real-time PCR. On Foxp1 a has an open reading frame 1 710 bp encoding 569 aa,while On Foxp1 b with 2 040 bp encoding 679 aa. Both of the putative On Foxp1 a / b contain a C2H2 zinc finger domain,a leucine zipper-like domain( V-x( 6)-L-x( 6)-L-x( 6)-L) and a winged-helix / fork head domain,which are typical motifs of Foxp subfamily. Compared with On Foxp1 a,On Foxp1 b shows a higher amino acid identity( 77. 4% versus 55. 0%) to mammalian Foxp1. Phylogenetic analysis reveals that On Foxp1 a / b together with the other known vertebrate Foxp1 form a different clade from Foxp2,Foxp3 and Foxp4.M eanwhile,On Foxp1 a / b separately form a fish-specific clade and On Foxp1 b is further clustered with Foxp1 from the higher vertebrates,which supports that On Foxp1 b has a closer relationship with mammalian Foxp1.The expressions of On Foxp1 a / b were detectable in all examined tissues. The highest mRNA expression of On Foxp1 a was observed in the testis,while On Foxp1 b in the heart. M oreover,the moderate expression of On Foxp1 b was also observed in the immune-related tissues,such as spleen,kidney,gills and intestine.Stimulation of peripheral blood mononuclear cells with PHA( 50 μg / m L) and PM A( 50 ng / m L) led to a significant increase of On Foxp1 b mRNA expression at 6,12 and 24 h,respectively,while no significant changes of On Foxp1 a mRNA expression were observed except stimulation of PHA at 24 h. These results imply that the homologous Foxp1 in lower vertebrate is involved in lymphocyte activation. After 24 h treatment of 6-month male( XY) tilapias with estrogen( 17-beta-estradiol,E2),the expressions of On Foxp1 a / b were significantly up-regulated in the intestine and kidney but not in the spleen. Taken together,our results suggest that both of On Foxp1 a / b coded by two different genes are the orthologues of mammals and have potential disproportionate functions. M oreover,the mRNA expression patterns of On Foxp1 a / b are related with the lymphocyte activation and regulated by E2 level in vivo. This study not only provides scientific data for the research on the molecular evolution of Foxp subfamily members in lower vertebrates,but also will promote further study of the fish molecular immune response.更多还原