Cloning and analysis of Actin from Paeonia lactiflora / 中草药

ABSTRACT

Objective: Genomic gene encoding Actin in Paeonia lactiflora was cloned in order to clarify the gene organization and its expression levels in different tissues in herbaceous peony. Methods: Based on cDNA sequences of Actin genes isolated from P. lactiflora reported by our laboratory, one pair of PCR primers was designed. PCR products of Actin genomic gene were successfully amplified with total genomic DNA extracted from herbecous peony cv. "Taohuafeixue" as template by high-fidelity DNA polymerase KOD-Plus, and then they were cloned to pMD18-T vector and be sequenced. The exons and introns of Actin gene were predicted with bioinformatic softwares. The homology was analyzed by Blastn at the nucleotide level and the molecular phylogenetic tree was constructed with MEGA5.0 software. Based on sequencing results, one pair of PCR primers was designed, and the expression levels of Actin gene in the roots, stems, flowers, and leaves in herbaceous peony were semi-quantified. Results: The sequencing results showed that Actin gene of herbaceous peony was 1405 bp length, which contained four exons and three introns. All the splicing sites in three introns were conservative at 5' donor with GU and 3' receptor with AG. It encoded 377 amino acids, and GenBank accession number was KF363830. A pair of PCR primers was designed and one of them was spaned the first intron, which could effectively prevent the false postive by genomic DNA contamination. The semi-quantitative RT-PCR analysis showed that the expression levels of Actin gene in the roots, stems, leaves, and flowers of herbaceous peony were almost constant. Conclusion: It is the first report on cloning and gene organization clarification of genomic gene encoding Actin in P. lactiflora. The semi-quantitative analyses indicate that Actin gene can be used as the internal standard gene for the expression analysis of the functional genes in herbaceous peony.