巨核酸酶I-SceI转基因元件的构建及其在斑马鱼中转基因效率的评估  

Construction of I-SceI transgenic elements and assessment of its transgenic efficiency in zebrafish

作　　者：孙成飞[1] 董浚键[1] 田园园[1] 余嘉欣 叶星[1,2] 

机构地区：[1]中国水产科学研究院珠江水产研究所农业部热带亚热带水产资源利用与养殖重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《上海海洋大学学报》2015年第5期650-655,共6页Journal of Shanghai Ocean University

基　　金：国家高技术研究发展计划(2011AA100404);国家自然科学基金面上项目(31272688)

摘　　要：巨核酸酶I-SceI是由酿酒酵母线粒体中核糖体大亚基上的Ⅰ型内含子编码的一种核酸内切酶,近年已被应用于转基因研究中。I-SceI介导的转基因大多是将I-SceI蛋白和带有其识别位点的质粒共注射于动物的受精卵,但注射酶蛋白前期处理耗时长且工作量大,直接用I-SceI的mR NA代替蛋白进行注射更为简捷。本研究应用I-SceI mR NA介导荧光蛋白基因在斑马鱼中的转植,进一步了解其转基因效率。首先设计合成带有巨核酸酶I-SceI编码序列的辅助质粒(pC S2-I-SceI),并分别构建了带有巨核酸酶I-SceI识别位点、EF1α启动子和荧光蛋白基因(eG FP/RFP)编码序列的供体质粒pI-SceI-EF1α-eG FP/RFP。辅助质粒pC S2-I-SceI体外转录成mR NA,以50 ng/μL供体质粒和100 ng/μL的I-SceI巨核酸酶mR NA共注射到斑马鱼(Danio rerio)受精卵中,注射后48 h的斑马鱼eG FP的平均荧光表达率可达75.86%。在斑马鱼的头部、躯干到尾部均可观察到荧光蛋白的表达,且随着胚胎发育,荧光信号逐渐增强。结果表明I-SceI系统在斑马鱼中可介导较高效率的转植,具有在经济鱼类转基因研究中应用的潜力。Meganuclease I-Sce I is an endonuclease,which is derived from the intron of mitochondrial 21 S r RNA gene of Saccharomyces cerevisiae and has been applied to transgenic study in recent years. Usually,to use this system,target plasmid and I-Sce I protein were microinjected together. However,preparation of the ISce I protein for microinjection is laborious and time consuming. Replacing the protein by I-Sce I m RNA would be more convenient. In this study,the transgene of fluorescence protein was mediated by I-Sce I m RNA to assess its transgenic efficiency in zebrafish. First,helper plasmid with I-Sce I coding sequence p CS2-I-Sce I was constructed and I-Sce I m RNA was transcripted in vitro. Donor plasmid,p I-Sce I-EF1α-e GFP and p I-Sce IEF1α-RFP donor plasmid with I-Sce I recognition sites, EF1α promoter and e GFP（ RFP） gene were constructed,and co-injected with I-Sce I m RNA into the 1-2 cell stage fertilized eggs of Danio rerio. The concentrations of donor plasmid and I-Sce I m RNA were 50 ng / μL and 100 ng / μL respectively. The e GFP fluorescence expression rate was 75. 86% in 48 h. The fluorescence can be observed in the head,body and tail of zebrafish and became stronger and stronger with the development of embryos. Our data suggest that ISce I can efficiently mediate gene transfer and has the potential to apply in the transgene of economic fish.

关 键 词：巨核酸酶 I-SCEI 斑马鱼 转基因 

分 类 号：S917[农业科学—水产科学]