斑马鱼HO1基因的表达特征及功能研究

STUDY ON EXPRESSION PATTERN AND FUNCTION OF ZEBRAFISH HO1

实验对血红素加氧酶1(HO1)在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%—86.8%,血红素结合标签相似性为87.5%—95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。

This paper has studied the function of heme oxygenase 1 （HO1） in the development of zebrafish. Zebrafish HO1 contains heme oxygenase domain, heme binding signature, five histidine residues and hydrophobic segment at carboxyl terminus. Analysis of the deduced amino acid sequences revealed zebrafish HO1 shared 44.1%-86.8%similarity with known sequences from other species. Analyzing embryos of different development stages by RT-PCR showed that zebrafish HO1 existed in unfertilized eggs. Its level increased from 24hpf and remained a high level expression until 72hpf. The tissue expression pattern analysis showed that zabrafish HO1 was expressed ubiquitously and the expression in brain, heart, gill and liver was obviously higher than others. Whole-mount in situ hybridization showed that HO1 transcript exists in yolk syncytial layer, blood and eyes during embyonic development. Using overexpression and knock down technology, we found that over-expression of HO1 had no apparent effect on the early development of zebrafish. Knocked down of HO1 gene by HO1 MO made the embryo development retarded. The abnormality and lethal rates of embryos after MO treatment were significantly increased compared with control. The level of IGF1 and IGFBP1 mRNA changed significantly after HO1 was knocked down. These results showed that HO1 can regulate of zebrafish embryonic development through IGF signaling.