建鲤内参基因β-actin的实时荧光定量PCR方法的建立

Establishment of a real time PCR assay for Cyprinus carpio var. jian β-actin as a reference gene

β-actin在真核细胞的生理过程中起着重要的作用,其表达范围广、表达量恒定,常作为内参基因应用于real time PCR中。本文通过RT-PCR克隆出建鲤(Cyprinus carpio var.jian)β-actin的部分cDNA序列,其长度为424 bp,翻译成138个氨基酸,计算的蛋白质分子量为15.4 ku。氨基酸同源性分析显示,建鲤β-actin与斑马鱼(Danio rerio)相似性最高,为99.3%。与其他鱼的相似性也较高,为97.8%～98.6%。同时也克隆出了建鲤β-actin相应的DNA序列,其长度为590 bp。cDNA和DNA的序列比对显示克隆出的建鲤β-actin含有2个内含子,设计一对跨越内含子的引物,采用SYBR GreenⅠ染料建立了real time PCR方法。以肝脏cDNA为标准品,建立了标准曲线,并进行了溶解曲线分析。结果表明,所建立的方法具有特异性强、相关系数高、线性范围广等优点,可应用于建鲤的功能基因表达研究。

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. fian β-actin with Danio rerio, Takifugu rubripes, Carassius auratus, etc. , showed that the homology were 99. 3% between C. carpio var. fian and D. rerio, however the amino acids homology was 97.8% -98.6% when comparing C. carpio var. fian with other fish. The corresponding DNA sequence of C. carpio var. fian 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 Green I 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.