The Internally Self-fertilizing Hermaphroditic Teleost Rivulus marmoratus (Cyprinodontiformes, Rivulidae) β-Actin Gene: Amplification and Sequence Analysis with Conserved Primers

Abstract:

To determine the ease and feasibility of amplifying the β-actin gene in fish by the polymerase chain reaction (PCR), genomic DNAs of several fish (Rivulus, Southern top mouth minnow, common fat minnow, oily bitterling, carp, Far Eastern catfish, medaka, and European flounder) were extracted and used as a template with conserved primers, designed on the basis of high amino acid homology (approximately 98% or more). Among them, the self-fertilizing hermaphroditic fish Rivulus marmoratus was chosen for further characterization. After amplification of the Rivulus β-actin PCR product with Taq polymerase, PCR product was subcloned to pCRII vector. After restriction enzyme mapping of Rivulus β-actin gene, the amplified insert was sequenced using ALF Express automatic DNA sequencer with conserved internal primers. The R. marmoratusβ-actin gene consists of 1763 bp encoding 375 amino acids including 5 exons and 4 introns. The splicing and acceptance sites of the exon and intron boundaries of the Rivulus β-actin gene were highly conserved with consensus sequences (GT/AG). The amino acid homology of R. marmoratusβ-actin to other species was high: 98.93% to human; 98.93%, Atlantic salmon; 98.93%, common carp; 98.93%, grass carp; 98.93%, zebrafish; 98.67%, medaka; and 98.40%, sea bream. To determine the expression of the R. marmoratusβ-actin gene in liver and ovary, reverse transcriptase–polymerase chain reaction was carried out with internal primers. In conclusion, these universal primers are successful in the rapid cloning of the fish β-actin gene by PCR, based on a high homology of the β-actin gene conserved through evolution. This approach will be applicable to the isolation of other β-actin homologues in the investigation of phylogenetic comparisons of fish species, along with a possible application to cloning strategy in other conserved genes.