建鲤内参基因EF-1α的实时荧光定量PCR方法的建立

真核生物延伸因子基因(EF-1α)在蛋白质翻译过程中起着重要的作用,其序列具有高度的保守性,常作为内参基因应用于real time PCR中。通过RT-PCR克隆出建鲤(Cyprinus carpio var.jian)EF-1α的部分cDNA序列,其长度为425 bp,翻译成140个氨基酸,Blast结果显示与其它鱼类leptin的相似性为98%。同时也克隆出建鲤EF-1α相应的DNA序列,共506 bp。cDNA和DNA的序列比对显示克隆出的建鲤EF-1α含有1个相位为0的内含子,在此基础上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,并进行了融解曲线分析。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。

Establishment of a Real Time PCR Assay for Cyprinus carpio var.jian EF-la As a Reference Gene

Eukaryotic elongation factor 1α(EF-1α) plays an important role in translation and its sequence is highly conservative as a housekeeping gene in real time PCR.The partial cDNA encoding EF-1αin Cyprinus carpio var.jian was isolated using RT-PCR.The sequence of cDNA was 425 bp in length encoding 140 amino acids residues,and the homology was about 98%between C.carpio var.jian and other fish using the Blast program.The DNA sequence of C.carpio var.jian EF-1αconsisting of 506 bp was also cloned.Comparing the partial cDNA to its genomic sequence revealed that C.carpio var.jian EF-1αgene consisted of a phase 0 intron. A pair of real time PCR primers cross intron was designed according to the intron sequence of EF-1αand a real time PCR method by SYBR GreenⅠwas established.The standard curve was established with liver cDNA as the standard template and the melting analysis was also carried out.The results showed that the real time PCR method for EF-1αhad the advantages of high specificity,good correlation coefficients and wide linear range,which supplied useful information for studying function gene expression.