Isolation of Tanichthys albonubes β actin gene and production of transgenic Tanichthys albonubes

A β actin cDNA of Tanichthys albonubes was isolated through the RT-PCR and RACE approach. The cDNA was 1,787-bp in length, including a 1,128-bp CDS, a 95-bp 5′UTR and a 564-bp 3′UTR. Genomic DNA containing the transcription region and 5′-flanking region was cloned based on the β actin cDNA by Genome walker. A 3,000-bp β actin gene promoter was then produced by PCR according to the sequences of the 5′-flanking region and the first intron. This promoter consisted of a 1,800-bp 5′-flanking region, and a 1,200-bp 5′-UTR. 3 transcription elements, CAAT box, CArG motif and TATA box were found in the 5′-flanking region. This promoter was inserted into the vector pDsRed2-1 and microinjected into fertilized eggs of Tanichthys albonubes to prove its transcription activity. The β actin promoter and GH CDS of Tanichthys albonubes were then fused to construct an expression vector pTLA-GH. GH-transgenic Tanichthys albonubes was obtained by microinjection of the pTLA-GH into the fertilized eggs . Fast-growth individuals were observed in the transgenic group and the body weight of the largest individual was 2.1-fold that of the maximum in its non-transgenic siblings in 100 dph. In addition, a co-injection strategy was employed with pTLA-DsRed and pTLA-GH vector and proven to enhance the efficiency of GH-transgenic fish detection.