斑马鱼Tg(Crystal-pro-gsdf-2A-egfp)质粒的构建及验证

Construction and validation of Tg( Crystal-pro-gsdf-2A-egfp) plasmid in zebrafish

为研究性腺体衍生因子基因(gsdf)在斑马鱼Danio rerio精巢发育过程中相关分子调控机制模型,提取了斑马鱼精巢总RNA,逆转录合成c DNA,并以其为模板,用RT-PCR法扩增得到gsdf基因,构建了以小鼠gamma-crystalin启动子启动的斑马鱼gsdf基因与增强型绿色荧光蛋白基因(egfp)融合的荧光素酶报告质粒Tg (Crystal-pro-gsdf-2A-egfp),采用显微注射技术注射该质粒至1细胞期的斑马鱼受精胚胎中。结果表明:在荧光显微镜下观察到19尾能在眼部晶状体中特异表达绿色荧光蛋白基因(gfp)的F0代斑马鱼,将表达了绿色荧光蛋白基因的斑马鱼同野生型(WT)非转基因斑马鱼杂交,最终获得能特异表达绿色荧光蛋白基因的F2代,且gsdf基因能在斑马鱼眼部晶状体中特异表达并稳定遗传。研究表明,在斑马鱼上建立的Tg (Crystal-pro-gsdf-2A-egfp)转基因品系,为研究和观察斑马鱼精巢发育的分子调控机制提供了新的方法。

Total RNA extracted from testis of zebrafish Danio rerio was reversely transcribed to cDNA as a template for amplification of gonadal soma-derived factor gene(gsdf)by RT-PCR,and a reporter vector to over-express gsdf-2A-egfp fusion protein was constructed under the mouse gamma-crystalin promoter in order to investigate the potential role of gsdf gene playing in the mechanism of testicular differentiation.This reporter(Tg:Crystal-pro-gsdf-2A-egfp)was microinjected into one cell stage of zebrafish embryos and enabled a green fluorescence protein gene(gfp)and gsdf expression in lens of the fish from embryos to adults,with both transgenic lines of zebrafish(F1).Among nineteen adults with gfp signals in eyes found under a fluorescence microscope,specifically expressed green fluorescent protein of F2generation was finally obtained by hybridization of zebrafish expressing green fluorescent protein with wild-type(WT)non-transgenic zebrafish.The findings indicate that gsdf-2A-egfp can be inheridated and stable passed into the next generation through germline transmission in zebrafish.The establishment of Tg(Crystal-pro-gsdf-2A-egfp)transgenic line in zebrafish and provided a new method for research and observation of molecular regulation mechanism of gsdf gene in the development of zebrafish testis.