Enhanced Expression of Genes Involved in the Biosynthesis Pathway of Tanshinones in Tetraploid Plants of Salvia Officinalis L.

Extended Abstract Introduction and Objective: Polyploidy is one of the main factors in plant adaptation that can increase secondary metabolites production in plants. Salvia officinalis L. is a perennial plant from the Lamiaceae family with a long history of use in the medicinal industry. Tanshinones are crucial active compounds biosynthesized in Salvia. This study was aimed to analyze the expression of genes involved in the tanshinones biosynthesis pathway in diploid and tetraploid sage plants and compare between them. Material and Methods: polyploidy in S. officinalis was induced by seed treatment with 0.5% colchicine for 24 h, and tetraploid plants were selected and confirmed by flow cytometry, chromosomal observations, morphological characteristics, and the number of stomata. RNA was extracted from the leaves samples of diploid and tetraploid plants of sage and then cDNA synthesis and the gene expression analysis of genes involved in the biosynthesis of tanshinones including KSL, IPPI, CMK, and DXR genes was performed using real-time PCR. In order to investigate the expression of genes by RT-PCR in sage leaf samples, the 18srRNA gene was used as a reference gene to normalize the data. The thermal program for amplification of the target genes by the RT-PCR method included the initial activation of the enzyme, the steps of denaturation, and binding of primers. The amplification accuracy of the genes product was confirmed by the melting curve of each gene and the amplification validity was checked by gel electrophoresis. Results: Evaluation of the quality of extracted RNAs with 1% agarose gel showed a relatively good quality of the extracted RNAs. The melting curves obtained from the RT-PCR reaction using forward and reverse primers for the target genes showed that the primers bind correctly to the target sites at the specified temperature and cause their specific amplification. The results of RT-PCR showed that the expression of the genes involved in the biosynthesis pathway of tanshinones including KSL, IPPI, CMK, and DXR genes in tetraploid plants was significantly increased compared with diploids. Conclusion: In tetraploid plants, the expression of genes involved in the biosynthesis of tanshinones increased and this could increase the production of these secondary metabolites. Analysis of gene expression in different polyploidy series can increase our knowledge of the molecular mechanisms of biosynthesis of secondary metabolites and improve their production through polyploidy induction.