作　　者： ; ; ; ; ; ; ;

机构地区： 中国水产科学研究院珠江水产研究所

出　　处： 《农业生物技术学报》 2008年第2期242-247,共6页

摘　　要： 采用高保真PCR技术从尼罗罗非鱼(Oreochromis niloticus)基因组中分离了β-肌动蛋白(β-actin)基因片段(GenBank登录号:EF026001)。序列分析显示该片段包括长为1643bpβ-actin基因5'端侧翼区的启动调控区和90bp的部分转录区序列。启动调控区包括一段长为108bpβ-actin基因上游调控序列、β-actin基因不翻译的第1个外显子和第1个内含子。上游调控序列中含有与转录活性密切相关的作用元件:CAAT box,TATA box和CArG box,分别位于转录起始位点上游的-92、-29和-62处。将尼罗罗非鱼β-actin基因的启动调控区定向克隆到不含启动子的红色荧光表达载体pDsRed2-1中,构建了重组荧光表达载体。重组载体经EcoRⅠ线性化后,采用显微注射技术将其注射到唐鱼(Tanichthys albonubes)的受精卵中,利用荧光显微镜观察外源基因增强型红色荧光蛋白基因(DsRed2)在唐鱼中的表达。结果表明,在荧光显微镜下以及普通解剖镜下均可观察到红色荧光,说明本实验分离到的罗非鱼β-actin基因启动子序列具有有效的驱动功能。 5＇-flanking region and partial open reading frame（ORF） of β-actin gene （GenBank accession No.EF026001） of Nile tilapia（Oreochromis niloticus） was cloned by PCR amplification. The segment included 1 643 bp regulatory sequence and 90 bp partial ORF which encoded a 30 amino acids peptide. The regulatory sequence contained 108 bp 5＇ proximal promoter,the first untranslated exon and the first intron of β-actin gene. The proximal promoter region contained elements that were critical for transcription activity, including the CCAAT box,TATA box and CArG box which located at -92,-29 and -62 bp upstream of transcription initiation site, respectively. The regulatory sequence was inserted into the promoterless pDsRed2-1 vector to construct expressing vector pNA-DsRed. The linearized pNA-DsRed was microinjected into the fertilized eggs of white cloud mountain minnow（Tanichthys albonubes）. The expression of DsRed2 gene in transgenic fish could be observed under microfluoroscope and anatomical lens. The results showed that the β-actin gene promoter possessed effective transcription activity.

关 键 词： 罗非鱼 肌动蛋白启动子 红色荧光蛋白 转基因 显微注射

领　　域： [农业科学]