摘要
环境雌激素严重危害人类的健康.为了简便直观地检测水环境中的雌激素污染,构建了一种转基因斑马鱼,在这种鱼的体内,利用卵黄蛋白原1(vitellogenin1,vtg1)的启动子调控报告基因绿色荧光蛋白(enhancedgreenfluorescentprotein,EGFP)的表达.用1ng/L的17!-炔雌醇(17'-ethynylestradiol,EE2)诱导4天后,仔鱼肝脏中出现绿色荧光.RT-PCR和整体原位杂交实验证实,仔鱼体内EGFP和vtg1的时空表达模式相同.通过在显微镜下观察转基因鱼的绿色荧光,可以直观判断水环境中是否含有雌激素活性物质.该研究为环境雌激素的监测提供了一种简便直观的新型工具.
Environmental estrogens （EEs） are synthetic chemical compounds and plant-derived substance that are capable of imitating the functions of natural in vivo estrogen hormones. It has been known that estrogenic endocrine disrupters, which can cause intersexuality and reproduction decline in animals and human, can also induce synthesis of female-specifc proteins such as vitellogenin （vtg） in male and juvenile fish. To establish a novel in vivo test system for rapidly detection of estrogenic chemicals, a transgenic zebrafish has been developed. First, the expression vector pvtg1-EGFP by ligating a 1.7 kb zebrafish vtgl promoter to a reporter gene EGFP （enhanced green fluorescent protein） was constructed. Next, the pvtgl-EGFP DNA was microinjected into one-cell stage embryos of the zebrafish. Then the embryos were raised and divided into three groups： group one （blank control） remained in embryo media; group two （solvent control） and group three were subject to continuous exposure of 0.001% alcohol and 1ng/L 17α-ethinylestradiol （EE2）, respectively. The three groups were observed for GFP expression with fluorescent microscopy every 12 h. Green fluorescence was first observed on day 5 in the livers of group three larvae, while it was observed neither in group one nor group two. Furthermore, RT-PCR and whole in situ hybridization experiments were performed and concurrent accumulation of vtgl and EGFP mRNAs in larvae after EE2 exposure were observed, which indicated that the expression of EGFP transgene was driven by the vtgl promoter. Last, by amplifying EGFP gene from the genomic DNA of fluorescent fish, it was verified that the EGFP transgene had been inserted into the genomic DNA of these fish. Through observing green fluorescence in these transgenic fish, the presence of estrogenic substance in the water can be easily detected. The transgenic zebrafish offers an easy to use tool for effective monitoring of environmental estrogens.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2006年第10期965-970,共6页
Progress In Biochemistry and Biophysics
基金
上海市自然科学基金重点项目(03DZ14020)~~
关键词
转基因斑马鱼
绿色荧光蛋白
卵黄蛋白原
启动子
环境雌激素
transgenic zebrafish, green fluorescent protein, vitellogenin, promoter, environmental estrogens
