斑马鱼4个小maf时空表达分析及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 SMALL MAF AND THEIR EFFECTS ON THE REGULATION OF ZYMOGENS IN ZEBRAFISH

Maf蛋白家族作为重要的转录调控因子,参与细胞众多生命进程。它分为大Maf蛋白和小Maf蛋白,小Maf蛋白需要与其他B-Zip家族蛋白结合形成异二聚体才能发挥作用。在斑马鱼(Danio rerio)中,小Maf包括Mafk、Mafg1、Mafg2和Maft,Mafg1和Mafg2是Mafg在鱼类中特有的分化出的两个旁系同源基因。为了研究这4个小maf在物种之间的进化关系及表达模式,实验对4个小maf进行进化树、染色体线性关系分析,结果显示4个小maf的分子进化方向与物种进化方向一致,mafk和maff在物种间的更趋于保守,mafg比前两者要相对活跃。同时,实验选取斑马鱼胚胎发育十个不同时期(1 cell、2 cells、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4个小maf中,mafg2的表达量最高,maft和mafk的表达量次之,mafg1的表达量最低。在胚胎发育的后期(48 hpf),除了mafg2在躯干的血液循环处有明显的表达,maft在躯干的血液循环处有较弱的表达,而除mafk和mafg1在血液循环处没有检测到表达之外,4个基因的表达部位基本重叠,都在全身广泛表达,这可能也与它们之间存在功能叠加和替换有关。利用双荧光素酶检测系统检测了4个小Maf参与调控胰外分泌酶原基因转录的不同功能,结果显示4个小maf的过表达都能使胰外分泌酶原基因的表达下调。研究结果将为深入研究4个小maf基因的功能叠加和互补作用奠定基础。

The members of musculoaponeurotic fibrosarcoma oncogene（Maf） family are important transcription factors that are involved in many cascades of cell biology. They are classified into large and small Maf proteins. The small Maf proteins of zebrafish include Mafk, Mafg1, Mafg2 and Maff（Maft）. Mafg1 and mafg2 are paraloges of mafg that only exist in fish genomes. The phylogenetic tree indicated that the evolution of the small Mafs was conservative. The systemic analyses of different small mafs also showed that mafk and maff were more conservative than mafg. In the present study we analyzed the spatial-temporal expression of these four mafs in the early development of zebrafish embryos using semiquantitative PCR and whole mount in situ hybridization. The expression of all four genes started at the one cell stage, suggesting that these genes were maternal and the expression were ubiquitous in early zebrafish embryos. Mafg2 showed the highest expression level among the four genes, and was specifically expressed in blood circulation after 48 hpf, together with maff. The results of dual-luciferase system also showed that the over expression of the four small Mafs led to down-regulation of the transcription of zymogens in a dose-dependent manner. Our study should contribute to better understanding of the functional superposition and complementarities of small Mafs.