斑马鱼nrf基因时空表达分析及其在调控胰外分泌酶原基因表达中的作用研究

THE SPATIAL-TEMPORAL EXPRESSION PATTERNS OF NRF AND THEIR DIFFERENT EFFECTS ON REGULATION OF ZYMOGENS IN ZEBRAFISH

通过GenBank搜索发现,斑马鱼中有6个nrf同源基因,即nfe2、nrf1a、nrf1b、nrf2a、nrf2b和nrf3。为了研究6个nrf在物种之间的关系,实验对6个Nrf蛋白进行系统发育树分析,结果显示6个Nrf蛋白的分子进化趋势与物种进化趋势一致,尽管在硬骨鱼类中由于基因组倍增的原因,nrf1和nrf2基因各产生了两个拷贝,但Nrf蛋白仍然是比较保守的。此外,实验选取斑马鱼胚胎发育十个不同时期(1 cell、2 cell、3.5 hpf、6 hpf、12 hpf、24 hpf、36 hpf、48 hpf、72 hpf、96 hpf),对其表达模式进行了详细的研究,结果显示:6个nrf在一细胞期都有转录信号,且胚胎发育早期(48 hpf之前)全身广泛表达。72 hpf后,nrf1b、nrf2a、nrf2b、nrf3和nfe2基因的表达部位主要集中在头部、部分躯干、肠处,nrf2b还在脊髓处有微弱的表达。总体来说,6个nrf基因的表达部位基本重叠。研究还利用双荧光素酶检测系统检测了6个Nrf蛋白对胰外分泌酶原基因(tryl)转录的调控,结果显示nrf1a、nrf2a、nrf2b与nfe2的过表达能使tryl的表达上调,而nrf1b与nrf3的过表达能抑制tryl的表达。研究结果将为深入研究斑马鱼6个nrf基因的功能叠加、互补及其功能歧化奠定基础。

The nuclear factor-erythroid-2（nfe2）-related factor（Nrf） regulates genes involving in antioxidative stress response, anti-inflammatory response, and detoxification of heavy metals. Six nrf genes（nfe2, nrf1 a, nrf1 b, nrf2 a, nrf2 b and nrf3） were found in zebrafish based on blasting of the Gen Bank. The phylogenetic tree analysis indicated that the vertebrate Nrf proteins were conserved during the evolution of species, in spite of the fact that the nrf1 and nrf2 respectively have two duplicates, resulted from the whole-genome duplication in teleosts. The results of whole mount in situ hybridization indicated that all 6 genes expressed at the one cell stage, and their expressions were ubiquitous in early stage of embryonic development. At 72 hpf, nrf1 b, nrf2 a, nrf2 b, nrf3 and nfe2 were specifically expressed in head, part of the trunk and gut, and nrf2 b was slightly expressed in the spinal cord. In general, the expressional sites of six nrf gene were overlap. Strikingly, over-expression of Nrf1 a, Nrf2 a, Nrf2 b and Nfe2 induced tryl expression, however Nrf1 b and Nrf3 overexpression reduced tryl expression with a dosage dependent pattern. These studies provide foundational data for functional superposition and complementarities among these six nrf genes.