Document Type: Research(Original) Article
Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
4-Coumarate CoA ligase (4CL) is one of the key enzymes in the phenylpropanoid pathway which converts cinnamic acid derivatives to active thioesters. Active thioesters are precursors of a vast number of secondary metabolites. Salvia macrosiphon as a valuable medicinal plant grows in Iran, Turkey and Afghanistan. This plant produces pharmacologically active metabolites including rosmarinic acid, flavonoids (apigenin, luteolin), sesquiterpenes and coumarins. Identification of genes encoding 4cl provides the opportunity to manipulate the biosynthetic pathways and mediate the carbon flux toward the corresponding metabolites. Mature wild type S. macrosiphon plants were collected in flowering and seed-bearing stages. In vitro cultures of S. macrosiphon were established on Murashige & Skoog (1/2 MS) medium. To increase the possibility of finding 4cl isoforms in the transcriptome, cultures were elicited by chitosan. Since the genomic sequence of S. macrosiphon was not available, degenerate and CODEHOP primers were designed based on the identified 4CL protein sequences. Using the CODEHOP primers two isoforms of putative 4cl genes have been identified in all organs of the wild type plant. Degenerate primers could only amplify the same genes from roots and seed-containing capsules. This might be due to a higher expression level of the genes in these organs. No 4cl isoform have been detected from cultures which might be due to the lower abundance of the transcript at early stages of in vitro plantlets. Phylogenetic analysis showed that the two isoforms of 4cl genes from S. macrosiphon and Salvia miltiorrhiza have been evolved from a common ancestor.