关键词: 建鲤, 内参基因, β-actin, 实时荧光定量PCR

Abstract: Eukaryotic β-actin played an important role in physiological process and expressed in almost all tissues at a constant expression level. It was always used as a reference gene in real time PCR. The partial cDNA encoding β-actin in Cyprinus carpio var. jian was isolated using RT-PCR. The length of cDNA was 424 bp encoding 138 amino acids with a calculated molecular weight of 15.4 ku. The comparison analysis of the deduced amino acid sequence of C. carpio var. jian β-actin with Danio rerio， Takifugu rubripes， Carassius auratus， etc.， showed that the homology were 99.3% between C. carpio var. jian and D. rerio， however the amino acids homology was 97.8%-98.6% when comparing C. carpio var. jian with other fish. The corresponding DNA sequence of C. carpio var. jian β-actin consisting of 590 bp was also cloned. Comparing the partial cDNA to its corresponding DNA revealed that C.carpio β\|actin gene consisted of two introns. A pair of real time PCR primers cross exon was designed according to the introns sequence of β-actin and a real time PCR method by SYBR GreenⅠ was established. The standard curve was established with liver cDNA as standard template and the melting analysis was also carried out. The results showed that the real time PCR method about β-actin had advantage of high specificity， good correlation coefficients and wide linear range， which supplied useful information in function gene expression study by real time PCR.

Key words: Cyprinus carpio var. jian, reference gene, β-actin, real time PCR