[Cloning of the promoter region of Leuciscus Merzbacheri beta-actin gene and detection of its function].
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 beta-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-N1-AFP III was destroyed by Aat II digestion. SZ21 regulatory sequence was inserted into the vector pEGFP-N1-AFP III (with destroyed CMV) that can express green fluorescence protein, and beta2 pEGFP-N1-AFP III recombination vector was constructed. Linearized beta2 pEGFP-N1-AFP III 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 beta-actin gene promoter SZ21 has the activity to switch on the EGFP expression in mammal cell, and has a con-tinued 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 beta-actin gene has effective transcription activity and can promote the expression of foreign gene.
Life-Science College, Shihezi University, Shihezi 832003, Xinjiang, China. hwg-t@163.com
Wen-Ge
WG
Hu
Feng-Xia
FX
Hao
Chuang-Fu
CF
Chen
Yuan-Zhi
YZ
Wang
Yan
Y
Ren
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000199:Actins