准噶尔雅罗鱼β-肌动蛋白基因启动子的克隆及其活性功能初步检测  

Cloning of the promoter region of Leuciscus Merzbacheri β-actin gene and detection of its function

作　　者：胡文革[1] 郝凤霞[1] 陈创夫[2] 王远志[3] 任艳[2] 

机构地区：[1]石河子大学生命科学学院,新疆石河子832003 [2]石河子大学动物科技学院,新疆石河子832003 [3]石河子大学医学院,新疆石河子832003

出　　处：《遗传》2009年第10期1029-1036,共8页Hereditas（Beijing)

基　　金：石河子大学科技发展计划项目(编号:ZRKX200744)资助

摘　　要：以开发利用新疆濒危鱼类准噶尔雅罗鱼(Leuciscus merzbacheri)基因资源为研究目的,利用PCR技术克隆准噶尔雅罗鱼的β-actin基因,得到的β-actin基因片段SZ21包含启动调控区,大小为2398bp。SZ21的启动调控区包括β-actin基因上游调控序列、第1、第2、第3和第4外显子部分序列。上游调控序列中含有对转录起重要作用的CAAT框、TATA框和CArG框等元件。对启动子序列在线分析表明,获得的启动子含有E-box、RU49、ZBPF、CEBP、CREB等多个重要转录因子结合位点。用AatⅡ破坏真核表达载体pEGFP-N1-AFPⅢ中的CMV启动子,将准噶尔雅罗鱼的SZ21启动调控区克隆到载体pEGFP-N1-AFPⅢ(CMV坏)上,构建成重组表达载体β2pEGFP-N1-AFPⅢ。脂质体转染BHK-21细胞。结果表明,克隆的准噶尔雅罗鱼β-actin基因启动子SZ21具有启动EGFP报告基因在哺乳动物细胞中表达的活性。通过BHK-21绿色荧光细胞的传代证实,克隆的启动子具有持续启动蛋白基因表达的活性,在细胞传代中可以遗传。PCR检测传代的BHK-21绿色荧光细胞基因组DNA,均能检测到SZ21目的片段。文章成功分离了具有活性功能的准噶尔雅罗鱼β-actin基因启动子。To utilize the gene resources of Leuciscus merzbacheri, a 2 398 bp （SZ21） DNA fragment including the 5′- flanking region and partial open reading frame of the β-actin gene was obtained through PCR amplification. SZ21 includes a regulatory sequence which contains the 5′-proximal promoter, the first, the second and the third exons and the partial fourth exon sequence. The 5′-proximal promoter region is critical for transcription activity including the CAAT box, TATA box and CArG box. The analysis of putative transcription binding sites of the promoter by on-line software revealed the presence of the critical transcription binding sites （such as E-box, RU49, ZBPF, CEBP and CREB）. CMV promoter for eukaryote vector pEGFP-N 1-AFPⅢ was destroyed by Aat Ⅱ digestion. SZ21 regulatory sequence was inserted into the vector pEGFP-N1-AFPⅢ （with destroyed CMV） that can express green fluorescence protein, and β2 pEGFP-N1-AFPⅢ recombi- nation vector was constructed. Linearized 132 pEGFP-N1-AFPⅢ was transfected into BHK-21 cell through lipofectin. EGFP expression Of the transgenic cell was observed by micro fluoroscope. The results indicated that the cloned Leuciscus merzbacheri β-actin gene promoter SZ21 has the activity to switch on the EGFP expression in mammal cell, and has a continued starting expression activity passing on from generation to generation in green fluorescence cell. In addition, the SZ21 target fragment was detected in the BHK-21 green fluorescence cell genomic DNA by PCR. This suggested that the SZ21 promoter of β-actin gene has effective transcription activity and can promote the expression of foreign gene.

关 键 词：准噶尔雅罗鱼 β-肌动蛋白启动子 脂质体转染 BHK-21细胞 活性功能 

分 类 号：S917.4[农业科学—水产科学]