斑马鱼外源基因acta1-AcGFP重组及其最佳注射浓度初探

Construction of Zebrafish acta1-AcGFP Recombinant and the Optimum Concentration for Micro-injection

肌动蛋白纤维(actin filament)又称微丝(microfilament,MF),由肌动蛋白(actin)组成,是骨骼肌的重要组成部分。通过PCR从斑马鱼基因组DNA中分离得到大小为2 019 bp的α肌动蛋白1(α-actin1)启动子所在区域片段。序列分析表明,该片段序列与NCBI公布的α-actin1基因5′侧翼区相应序列的相似性为98.7%,并且含有多个MEF2,E-box等对肌肉表达起调控作用的顺式元件以及在转录中起重要作用的TATA-box。将该启动子片段与绿色荧光蛋白基因(AcGFP)重组,构建肌动蛋白特异性表达载体。采用显微注射法,将线性化表达载体注入斑马鱼受精卵中,获得转绿色荧光蛋白基因斑马鱼,平均表达率最高为16.5%。并初步确定400 ng/μl为外源基因(acta1-AcGFP)的最佳注射浓度。通过观察发现,AcGFP基因主要在骨骼肌中表达,且表达量随仔鱼肌肉的发育呈增长趋势。证明了分离得到的α-actin1启动子所在区域片段具有有效的驱动功能,为进一步开展转基因观赏鱼的研究与开发提供了依据。

In this study, 2 019 bp size of α-actinl promoter region fragment was obtained by PCR method from zebrafish （Danio rerio） genomic DNA. Sequence analysis showed the similarity between the promoter region obtained and the corresponding 5 flanking region of α-actinl gene published on NCBI was 98.7%. The promoter region contains a number of MEF2, E-box and some other muscle control ciselements as well as TATA-box which plays an important role in transcription. This promoter region fragment was then ligated in green fluorescent protein gene （AcGFP） vector to construct actin-specific expression vector which was then injected into zebrafish eggs by the method of micro-injection. The average expression level was as high as 16. 5% and the optimum concentration of ae- tal-AcGFP gene for micro-injection was set as 400 mg/μl. The AcGFP was mainly expressed in skeletal muscle and the fluorescent spread as the development of the fry＇s muscle. This study proved the α-actinl promoter region fragments separated have a valid driver＇s features, and also provided a useful recombinant for transgenic fish research.