clGH与EGFP基因融合表达载体的构建及其在斑马鱼胚胎发育阶段的表达

Construction of a Fusion Expression Vector Containing GH of Clarias lazera and EGFP Gene and Its Expression at Embryonic Developmental Stage of Danio rerio

以模式动物斑马鱼(Danio rerio)为材料,研究了革胡子鲶生长激素(growth hormone of clarias lazera,clGH)基因的表达。以clGH基因为外源基因,以增强型荧光蛋白(enhanced green fluorescent protein,EGFP)基因为报告基因,通过体外酶切和连接,构建了含有这两个基因的融合表达载体pCEGFP-clGH。通过显微注射方法,将pCEGFP-clGH转入斑马鱼受精卵进行表达。结果显示,成功得到了携带外源clGH基因的转基因斑马鱼,并可在胚胎发育的不同阶段(0-72h)观察到绿色荧光,荧光表达率分别为7.2%,45.7%,71.7%和8.5%;外源基因的表达特点为嵌合型、瞬时表达;外源基因最早在斑马鱼胚胎发育的囊胚期(3h)开始表达,在注射后24h达到最强,在仔鱼孵出前后(72h)表达水平降至最低。本试验构建的融合表达载体正确,为今后进一步研究clGH基因的功能及利用clGH基因进行快速生长转基因鱼的培育提供参考。

In this research,the expression of clGH gene was studied in model animal zebrafish（Danio rerio）.The fusion expression vector pCEGFP-clGH,containing target gene clGH（growth hormone of Clarias lazera,clGH）and EGFP（enhanced green fluorescent protein,EGFP）as reporter gene was constructed,and transfered into fertilized eggs of zebrafish by microinjection and expressed.Results showed that transgenic embryos/juveniles of zebrafish carrying clGH gene was successfully obtained and identified by PCR and RT-PCR.At the different stage of embryonic development of zebrafish（0-72 h after injection）,successive green fluorescent could be observed under microscope,with GFP expression rate of 7.2%,45.7%,71.7%and 8.5%,respectively.GFP expression pattern was mosaic and transient,which showed that expression of clGH gene started at blastula stage of embryonic development,approached its highest level at 24 h after injection,and declined from 24 h to 72 h after injection.Thus,the fusion expression vector was constructed correctly,and results obtained could establish satisfactory foundation for further study on the fuction of clGH and production of fast-growth fish varieties.