Nucleotide sequence and mRNA expression analysis of β-actin gene in the orange-spotted grouper Epinephelus coioides

The full-length cDNA, encoding the orange-spotted grouper beta-actin and spanning 1920 bp including a poly (A) tail, was cloned from its brain cDNA library. The open reading frame encodes a protein of 375 amino acids. Sequence analysis indicated that it contained the typical structural features of cytoplasmic actins, and showed higher homology with other vertebrate beta-actin than any other members of the actin family. The partial genomic sequence indicated that the organization of the beta-actin gene in the orange-spotted grouper might also be conserved. Northern blot analysis indicated that it was expressed at high levels in the brain, spleen, adipose tissue, ovary, and liver, but at low levels in the gill filament and heart, and at a very low level in the kidney. The expression of beta-actin gene in the skeletal muscle was barely detectable. These results indicated that the expression of the orange-spotted grouper beta-actin gene showed significant variation in different tissues. Therefore, caution should be taken when using beta-actin gene as an internal control in the normalization of gene expression among tissues. Whereas, semi-quantitative RT-PCR analysis indicated that treatment with 17 alpha-methyltestosterone (MT) had little effect on the mRNA expression of beta-actin gene in the in vitro incubated hypothalamus, pituitary, and ovary fragments of the orange-spotted grouper, suggesting beta-actin can be used as an internal control for RT-PCR analysis of MT effects on gene expression in these tissues.