微囊藻毒素去毒酶转基因模型的构建

根据已克隆的鲢鱼(Hypophthalmichthysmolitrix)微囊藻毒素去毒酶cDNA全序列设计特异引物,利用PCR方法获得鲢鱼微囊藻毒素去毒酶基因编码区,将该编码区与绿色荧光蛋白连接,分别构建融合表达载体pEGFP-N1-sGST和双顺反子表达载体pIRES2-EGFP-sGST。利用脂质体转染法将融合表达载体pEGFP-N1-sGST转染Hela细胞,60h后检测到绿色荧光基因表达;通过显微注射,将双顺反子表达载体pIRES2-EGFP-sGST注入斑马鱼(Daniorerio)受精卵,获得了转鲢鱼微囊藻毒素去毒酶基因斑马鱼,从而构建了微囊藻毒素去毒酶转基因模型。上述2种转基因模型的成功构建为进一步研究鲢鱼、鳙鱼(Aristichthysnobilis)、罗非鱼(Oreochromisnilotica)等淡水鱼类微囊藻毒素去毒酶基因调控元件、去毒分子机理及研发转基因鲢鱼、鳙鱼、罗非鱼等微囊藻毒素高效生物去毒器奠定了基础。

Construction of microsystin-detoxifizyme transgenic model

cDNA fragment encoding complete sequence of silver carp(Hypophthalmichthys molitrix) microcystin-deto-xifizyme was subcloned to construct a fusing gene expression vector pEGFP-N1-sGST,and a dicistronic expression vector pIRES2-EGFP-sGST.Using lipofectin method,the fusing gene expression vector pEGFP-N1-sGST was transfected into Hela cells.The emission of fluorescence was observed after 60 hours.Through microinjection,the dicistronic expression vector pIRES2-EGFP-sGST was delivered into embryos of zebrafish(Danio rerio),and the silver carp microcystin-detoxifizyme transgenic zebrafish was obtained.The success of the two transgenic models makes it possible to further analyze the cis-regulatory elements of the microcystin-detoxifizyme gene,and to develop a strain of transgenic planktivorous fish,used as a high-efficient biodetoxicator for microcystin in lakes and reservoirs.