Molecular Cloning And Structure-functional Analysis Of The β-actin Gene In Chinese Perch (Siniperca Chuatsi) And Other Four Freshwater Fishes

β-actin is a member of the actin family found in all eukaryotic non-muscle cells. It plays a key role in maintaining cytoskeletal structure, cell motility, cell division, intracellular movements and contractile processes. As a kind of cytoplasmic actins, the expression of β-actin in all tissues is correspondingly stable. Based on this characteristic, it is usually used as control gene in quantitative or semi-quantitative research. β-actin is a housekeeper gene, and has a potent promoter which can improve transgene expression level efficiently without the limitation of tissue. This advantage makes β-actin promoter play an important role in the research of transgenic animals. Chinese perch (Siniperca chuatsi) has high economic value in China, whereas bighead carp (Aristichthys nobilis), catfish (Clarias fuscus), mud carp (Cirrhinus molitorella) and Taiwan snakehead (Channa maculata) are the major Chinese cultivated freshwater fishes. In this study, we used RT-PCR and RACE methods to get the full-length cDNA of Chinese perch β-actin gene. Partial cDNA sequences of bighead carp, catfish, mud carp and Taiwan snakehead β-actin gene were also obtained. Then, we cloned 5'-flanking region of Chinese perch β-actin gene using genome walker method. The goal of this research is to provide reliable basic data for quantitative RT-PCR, to improve the validity and specialty of external standard primer design, and to build the theoretic base of transgenic research.Fresh Chinese perch, bighead carp, catfish, mud carp and Taiwan snakehead for the research were bought from the market, and then their livers were separated for RNA isolation. According to the deduced amino acid sequences of β-actin from other vertebrates, three degenerate primers were designed and synthesized for cloning the cDNA of each gene by RT-PCR. The full-length β-actin cDNA sequence of Chinese perch was further obtained by using the method of 3'- and 5'- rapid amplification of cDNA ends (RACE). Genome walker method was applied to get 5'-flanking region of Chinese perch β-actin gene. The full-length Chinese perch β-actin cDNA obtained was determined to be 1897bp in length with an open reading frame (ORF) that encodes 375 amino acids. Partial cDNA sequences of bighead carp, catfish, mud carp...