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.
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.