诺丽Actin基因片段克隆及实时荧光定量PCR方法的建立

摘要

In order to solve the problem of the lack of reference control gene in real-time fluorescence quantitative PCR (qRT-PCR) of noni,the degenerate primers were designed based on the conserved sequences of Actin genes from the other plants and the cDNA was reverse-transcribed from total RNA as template and amplified using PCR,and the gene fragment was cloned into pMD-18T vector.The positive clone was sequenced after being identified by PCR.The sequencing results revealed that the fragment ofActin gene from noni contained 393 bp,encoding a protein of 131 amino acids.Homology comparison with other plants actin sequences in GenBank showed that it shared the highest identities (96％) and positives (98％) with actin 1 of Vaccaria hispanica.The qRT-PCR indicated that the noni Actin gene was stable expression in various organs and in different fruit development periods,and the expression levels were basically consistent,so it was suitable as a reference control gene for the analysis ofnoni gene expression.%为了解决诺丽实时荧光定量PCR(qRT-PCR)检测中无内参基因的现状,根据其他植物Actin基因的保守序列设计一对简并性引物,以诺丽果实总RNA反转录成cDNA为模板,进行PCR扩增,扩增出的基因片段克隆到pMD-18T载体,阳性克隆经PCR鉴定后测序.序列结果表明:该片段长393 bp,编码131个氨基酸;所得序列与麦蓝菜actin 1有最高的一致性(96％)和相似性(98％).qRT-PCR结果表明,诺丽Actin基因在诺丽的各个组织、果实不同发育时期都能稳定表达,且表达水平基本一致,适合在诺丽基因表达研究中作为内参基因.