稀有鮈鲫β-肌动蛋白启动子的克隆及其驱动活性的初步检测

Cloning of β-actin promoter of Gobiocypris rarus and detection of its transcription activity

稀有鮈鲫是一种新型的模式实验鱼,具有利用转基因技术培育开发成为观赏性鱼类的潜在可能。本实验采用PCR方法,从稀有鮈鲫基因组DNA中分离得到大小为1584bp的β肌动蛋白(β-actin)基因片段。序列分析表明,该片段包括长为1507bp的启动调控区和77bp的部分转录区序列。启动调控区包括一段长为109bpβ-actin基因上游调控序列,不翻译的外显子1和内含子1。上游调控序列中含有TATAbox,CAATbox和CArGbox等与转录活性密切相关的作用元件,并且在稀有鮈鲫β-actin基因第一个内含子中也含有1个CArG调控元件。将该启动子片段克隆到绿色荧光表达载体(pAcGFP1-1)上,显微注射入稀有鮈鲫的受精卵中,通过荧光显微镜能观察到绿色荧光蛋白的表达。本实验成功分离了具有驱动活性的稀有鮈鲫β-actin基因启动子。

As a new laboratory fish in China,Gobiocypris rarus has the potential to develop into an ornamental fish by use of the transgenic techniques. A DNA fragment of 1,584bp of β-actin gene from G. rarus genome was obtained by PCR,which included a 1,507bp of a regulatory region and a 77bp of partial open reading frame （ORF）. The regulatory region included a 109bp of 5′ proximal promoter,an untranslated exon, and an intron of β-actin gene. The proximal promoter region contained consensus sequences of TATA box,CCAAT box and CArG box which were critical for transcription activity. Moreover,the intron also contained a typical CArG box. This promoter region fragment was inserted into green fluorescent protein gene vector （pAcGFPI-1） ,and the recombinant is then injected into the fertilized eggs of G. rarus by the method of micro-injection. The green fluorescence was observed under micro fluoroscope. This study proves that the cloned β-actin promoter has effective transcription activity.