Characterization of large yellow croaker ( Pseudosciaena crocea) β-actin promoter supports β-actin gene as an internal control for gene expression modulation and its potential application in transgenic studies in fish

Abstract

As a housekeeping gene, β-actin is one of the most commonly used reference gene and its promoter is widely used in transgenic studies in mammals and fish. In this study, we used genomic walker technology to clone the β-actin gene ( Lycβ-actin) promoter sequence from large yellow croaker, an economically important marine fish in China. The Lycβ-actin promoter region spans 3350 nucleotides (nt) and contains several transcription factor binding sites and a conserved enhancer motif (ATGGTAATAA) in the first intron. A promoter activity assay showed that this promoter region can drive enhanced green fluorescent protein (EGFP) gene expression in the fish cell line, EPC. Luciferase activity analysis demonstrated that the activity of the Lycβ-actin promoter is not affected by poly(I:C) or lipopolysaccharide (LPS) stimulation. Absolute real-time PCR analysis of various tissues revealed that Lycβ-actin expression levels are not significantly altered by poly(I:C) or inactivated trivalent bacterial vaccine ( P > 0.05). These results suggest that β-actin can be used as a suitable internal control for gene expression modulation in response to immune stimulations in large yellow croaker. In vivo transgenic experiments showed that the Lycβ-actin promoter region can drive efficient EGFP expression in large yellow croaker fries or fertilized zebrafish eggs, supporting its potential application in transgenic studies in fish.