香樟Actin基因的克隆及表达分析

摘要

As a house-keeping gene,Actin has been always being used as an internal standard to normalize mRNA levels between different samples by quantitative real-time PCR, thus plays an important role in gene expression analysis. In this research, through a method of homology cloning, a pair of degenerate primers were designed based on the conserved sequences of Actin genes from other plants submitted to GenBank, and then 4 cDNA fragments from Cinnamomum camphora were obtained using RT-PCR. Molecular biological analysis showed that, each of the 4 cDNA fragments was 998 bp and encoded a putative protein of 332 amino acids. Moreover, according to the homology analysis, the 4 cDNA fragments belonged to Actin subfamily, and they were named CcACTa,CcACTb,CcACTc and CcACTd, and deposited in GenBank (Accession number:KM086736 KM086737 KM086738 and KM086739). Quantitative real-time PCR results revealed that the expression level of CcACTc in different organs such as root, stem, leaf and leaves under low temperature treaments was relatively stable. It could serve as a candidate reference gene.%Actin作为一个看家基因,经常在实时定量PCR中被用作内参对不同样品中的mRNA进行定量,因此在基因表达分析中扮演重要的角色.利用同源克隆的方法,根据GenBank中已经公布的其他植物的Actin基因的保守序列设计简并引物,采用RT-PCR(Reverse Transcription Polymerase Chain Reaction)技术从香樟中获得了4个cDNA片段.分子生物学分析软件分析结果显示,4个片段大小为均为998 bp,编码332个氨基酸;同源性分析表明,所获得的片段属于Actin亚家族,分别命名为CcACTa、CcACTb、CcACTc和CcACTd并提交GenBank(登录号:KM086736、KM086737、KM086738和KM086739).实时定量PCR结果显示, CcACTc在根、茎、叶及低温处理的叶片中表达量相对稳定,可以作为候选内参基因.