Cloning and expression analysis on Actin gene from Paeonia lactiflora / 中草药

ABSTRACT

Objective: Cloning of Actin gene from Paeonia lactiflora and expression analysis of Actin gene with RT-PCR technique. Methods: Based on cDNA sequences of Actin genes isolated from the related species reported in GenBank, one pair of PCR primers was designed. PCR products of Actin gene were successfully amplified with RT-PCR technique using cDNA synthesized from the total RNA extracted from the roots of P. lactiflora 'Taohuafeixue' as template, and then they were cloned to pMD18-T vector with TA cloning method. The positive clones which were characterized with colony PCR, plasmid PCR, and enzyme digestion method were sequenced. Based on the sequencing results, one pair of PCR primers was designed, and the expression profile of Actin gene in the roots, stems, flowers, and leaves in P. lactiflora were semi-quantified with RT-PCR technique. Results: The sequencing results showed that the length of Actin gene of P. lactiflora was 1134 bp, encoding 377 amino acids, whose GenBank accession number was JX310002. Sequence analysis showed that Actin of P. lactiflora contained three kinds of characteristic signal sequences, whose homologous similarity in amino acid level with other plants was up to 99%. Based on homology modeling analysis, it revealed that there were four domains in its 3D structure. Bioinformatic software predicted that the molecular weight of Actin of herbaceous peony was 4.17 × 104, and the isoelectric point (pI) was 5.31. It was a hydrophilic and stable protein which was located in cytoplasm, without the transmembrane domain or signal peptide. Semi-quantitative RT-PCR analysis showed that the gene expression levels of Actin gene in the roots, stems, flowers, and leaves of P. lactiflora were almost constant. Conclusion: It is the first report on the cloning of Actin gene from P. lactiflora and the semi-quantitative analysis indicates that Actin gene can be used as the internal standard gene for the expression analysis of functional genes in P. lactiflora. This project plays a base for the effective application of Actin gene in P. lactiflora.