Cloning, sequencing and expression analysis of C4H gene in Ferula pseudalliacea

Ferula pseudalliacea is a perennial and monocarpic plant species belonging to Apiace-ae family. This species is distributed from Middle Asia to Northern Europe. The main secondary me-tabolites present in Ferula genus comprise coumarins, sesquiterpene coumarins and di-sesquiterpene coumarins. Based on phytochemicals present in this plant species, it has been extensively utilized by local people in traditional medicine for long time. Phenylpropanoid pathway is mainly responsible for biosynthesis of these valuable phytochemicals. Cynamate-4-hydrolxylase (C4H) is one of the main and key enzyme in phenylpropanoid pathway. To date, there is no genomic or transcriptomic information in Ferula pseudalliacea and as the first report, we aimed to clone and sequence C4H and its expression analysis in root, stem, leaf, inflorescence and immature seed in this valuable medicinal plant species.

Different organs of F. pseudalliacea were collected from Gazne village near Sanandaj city in June 2019. RNA was extracted from collected organs using LiCl method. cDNA was synthesized using Yekta Tajihz Azmia kit in 20 µl reactions. PCR products amplified with pri-mer designed from conserved regions of C4H among apiaceous species and it was then cloned within pTG19. The verified recombinant plasmid were send for Applied Biosystem company for sequenc-ing. To confirm and asses the expression of C4H, both Real-Time PCR and semi-quantitative PCR were employed. The phylogenetic tree of C4H was obtained using Omega software based on the Neighbor-Joining method with bootstrap 1000 for amino acid sequence.

The obtained sequences from C4H clones was aligned and verified using BlastX. In the re-sulted dendrogram, C4H sequence from F. pseudalliacea was grouped with C4H gene from other species within Apiaceae family showing their clos relationship. The highest sequence identity was observed with C4H from Daucus carota which was fallen in same cluster with C4H from F. pseudal-liacea. It is shown that C4H has 7 motifs one of this motifs belongs to conserved motif cytochrome-cysteine 450 with FGVGRRSCPG sequence.

As the seconf most important gene in phenylpropanoid biosynthetic pathway, C4H plays an undeniable role in production of phenolic phytochemicals in particular in inflorescence which had the highest C4H expression rate. Studies performed on the C4H expression in other spe-cies revealed a specific action of C4H in biosynthesis of special secondary metabolites in specific tis-sue. As the phenolic compounds encompass a diverse range of secondary metabolites in forms of volatile and extracts, further investigations are needed to understand the exact role of C4H enzyme. Moreover, the identified gene sequence can be sued in production of recombinant constituents in other species which have the corresponding precursors.