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

Abstract:

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 eu-karyote vector pEGFP-N1-AFPⅢ was destroyed by AatⅡdigestion. 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 β2 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.