Karakterisasi Promoter β-Actin Ikan Nila (Oreochromis Niloticus)

Abstract

Promoter is one of the factors determining the successful of transgenesis. In this study we isolated and characterized β-actin promoter from Nile tilapia (tiBP) towards production of autotransgenic tilapia. β-actin promoter has high activity in muscle. Sequence of tiBP promoter was isolated by using PCR method. Sequencing was performed using ABI PRISM 3100 machine. Analysis of sequences was conducted using GENETYX version 7 and TFBind softwares. DNA fragment of PCR amplification product digested from the vector cloning was then ligated with pEGFP-N1 to generate ptiBP-EGFP construct. The construct was microinjected into one-cell stage of zebrafish (Danio rerio) embryos to test the tiBP promoter activity. EGFP gene expression was observed by fluorescence microscope. The result of sequence analysis showed that the length of DNA fragment obtained is about 1.5 kb and containing the evolutionary conserved sequences of transcription factor for β-actin promoter including CCAAT, CArG and TATA boxes. Furthermore, tiBP sequence in ptiBP-EGFP construct could regulated GFP expression in muscle of zebrafish embryos injected with the construct. The results suggested that PCR amplification product is the regulator sequence of tilapia β-actin gene. Autotransgenic tilapia can be then produced by changing GFP gene fragment of ptiBP-EGFP construct with genes from tilapia encoding important traits in aquaculture.