Through PCR amplification, 1.3 kb of 5'-proximal promoter (TA, 1.3 kb) of the beta-actin gene of white cloud mountain minnow Tanichthys albonubes was obtained. Using Genome Walker, a 1.7 kb 5'-upstream sequence from the proximal promoter of the beta-actin gene was isolated, and a further promoter (3.0 kb in size) was amplified according to the isolated 5'-proximal and upstream sequences (TLA, 3.0 kb). Both the 1.3 kb and 3.0 kb promoters contain elements that were critical to the transcription activity of other species, including the CCAAT Box (-89 approximately -85), CArG Box (-59 approximately -49), TATA Box (-26 approximately -20). Results of putative transcription binding sites analysis of the promoters by software TRANSFAC 6.0 revealed the presence of E-box, several transcript binding sites NF-Y, SP1 (Stimulating Protein 1), AP1 (Activator Protein 1), and some more transcription binding sites existing in the further promoter. The two promoter sequences were inserted into the expression vector to construct the recombinant expression vector, pTA-DsRed and pTLA-DsRed, respectively. The vectors were microinjected into the fertilized eggs of Tanichthys albonubes and higher positive rate was obtained and stronger red fluorescence was observed in pTLA-DsRed transgenic fish. RT-PCR analysis showed that RFP (Red fluorescent protein) mRNA level in pTLA-DsRed transgenic fish was 35.7% higher than that of the pTA-DsRed transgenic fish of 15-days-post-hatched. The present study showed that both the proximal and further promoter sequences have effective transcription activities and the 3.0 kb promoter possesses higher potent activity than that of the 1.3 kb promoter.
5' Untranslated Regions ; genetics ; Actins ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Cyprinidae ; genetics ; Genetic Vectors ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism